goat anti rabbit igg  (Jackson Immuno)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    AffiniPure Goat Anti Rabbit IgG F ab 2 fragment specific
    Description:
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with the F ab 2 Fab portion of rabbit IgG It also reacts with the light chains of other rabbit immunoglobulins No antibody was detected against the Fc portion of rabbit IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    Catalog Number:
    111-005-006
    Price:
    92.0
    Category:
    Whole IgG Affinity Purified Antibodies
    Conjugate:
    Unconjugated
    Size:
    2 0 mg
    Format:
    Whole IgG
    Host:
    Goat
    Buy from Supplier


    Structured Review

    Jackson Immuno goat anti rabbit igg
    (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and <t>FITC-conjugated</t> goat anti–rabbit <t>IgG,</t> and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with the F ab 2 Fab portion of rabbit IgG It also reacts with the light chains of other rabbit immunoglobulins No antibody was detected against the Fc portion of rabbit IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    https://www.bioz.com/result/goat anti rabbit igg/product/Jackson Immuno
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis"

    Article Title: Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20050433

    (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.
    Figure Legend Snippet: (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.

    Techniques Used: Staining, Incubation, Microscopy, Translocation Assay, Fractionation, Western Blot, Marker

    (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.
    Figure Legend Snippet: (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Activation Assay, Staining, Expressing, Fluorescence

    2) Product Images from "Aminopeptidase N (CD13) Is Involved in Phagocytic Processes in Human Dendritic Cells and Macrophages"

    Article Title: Aminopeptidase N (CD13) Is Involved in Phagocytic Processes in Human Dendritic Cells and Macrophages

    Journal: BioMed Research International

    doi: 10.1155/2013/562984

    CD13 localizes at the phagosomal membrane after phagocytosis of zymosan particles in human monocyte-derived macrophages. Human MD-Ms were incubated with Texas Red-labeled zymosan particles for 30 min at 37°C. After washing away noningested particles, cells were fixed, permeabilized, and stained with anti-CD13 antibody and a secondary FITC-labeled goat anti-murine IgG. Samples were analyzed using confocal microscopy. From left to right: images of the same cell taken at 0.75 μ m intervals in the z axis. Bar: 5.0 μ m.
    Figure Legend Snippet: CD13 localizes at the phagosomal membrane after phagocytosis of zymosan particles in human monocyte-derived macrophages. Human MD-Ms were incubated with Texas Red-labeled zymosan particles for 30 min at 37°C. After washing away noningested particles, cells were fixed, permeabilized, and stained with anti-CD13 antibody and a secondary FITC-labeled goat anti-murine IgG. Samples were analyzed using confocal microscopy. From left to right: images of the same cell taken at 0.75 μ m intervals in the z axis. Bar: 5.0 μ m.

    Techniques Used: Derivative Assay, Mass Spectrometry, Incubation, Labeling, Staining, Confocal Microscopy

    3) Product Images from "Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1"

    Article Title: Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

    Journal: Journal of Virology

    doi: 10.1128/JVI.03737-14

    Binding of mouse monoclonal antibodies to HKU1 S. (A) IFA. HEK 293T cells expressing HKU1 S protein were fixed and stained with the indicated MAbs (undiluted hybridoma supernatants), followed by FITC-conjugated goat anti-mouse IgG. The concentration of
    Figure Legend Snippet: Binding of mouse monoclonal antibodies to HKU1 S. (A) IFA. HEK 293T cells expressing HKU1 S protein were fixed and stained with the indicated MAbs (undiluted hybridoma supernatants), followed by FITC-conjugated goat anti-mouse IgG. The concentration of

    Techniques Used: Binding Assay, Immunofluorescence, Expressing, Staining, Concentration Assay

    4) Product Images from "Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration"

    Article Title: Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20150354

    SAC is critical for leukocyte transmigration in vivo. (a) Validation of rat anti–mouse CD99 monoclonal antibody, clone 3F11. Flow cytometry analysis of control (shaded) or anti–mouse CD99 (3F11, thick line) mAb binding to parental or mouse CD99-transfected Ba/F3 cells. (b) The ears of wild-type FVB/n mice (age and sex matched littermates) pretreated with DMSO, anti-CD99 (3F11 mAb), or KH7 (5 µmol/kg) were stimulated with croton oil (1%, 20 µl/ear) or carrier (10% olive oil/90% acetone). After 5 h, mice were sacrificed, their ears were harvested, and immunohistochemical staining was performed using anti-PECAM (ECs), anti-MRP14 (neutrophils), and anti–collagen-IV (basement membrane). 3D confocal images were acquired for each sample. (c) Quantification of results above. Percent of leukocytes extravasated within 50 µm of venules per field of view. (d) Model for quantification of site of arrest. Neutrophils were scored as being in one of six positions: luminal (1), apically arrested (2), arrested partway through the endothelium (3), arrested on the basement membrane (4), migrating through the basement membrane (5), or fully extravasated (6). (e) Quantification of the site of arrest for anti-CD99 and KH7-treated animals. (f) The ears of wild-type FVB/n mice (age- and sex-matched littermates) pretreated with anti-CD99 or rat IgG (control) were stimulated with croton oil. After 3 h, mice received dimaprit (10 mg drug/kg animal) or carrier (H 2 O). Mice were sacrificed 2 h later, their tissue stained, and analyzed as described in panel b. (g) Quantification of results. Percent of leukocytes extravasated within 50 µm of venules per field of view. (h) Additional mice pretreated with anti-CD99 mAb were sacrificed at the time dimaprit was given (3 h) to ensure that the anti-CD99 blockade was present throughout the experiments (5 h total). (i) Quantification of the site of arrest for anti-CD99/carrier and anti-CD99/dimaprit-treated animals. 100–200 cells were analyzed per ear. Total PMN per field of view, vessel length, and vessel diameter were equivalent for all conditions tested (not depicted). Images were acquired with a 40× objective ( n = 1.00). Insets show xz-orthogonal view (where yellow bar dissects the vessel) to demonstrate site of neutrophil arrest. Bars, 25 µm. Two to three mice per condition were used for each experiment. Images are representative of two (f) or three (a and b) independent experiments. Data represent the average value of two (g-i) or three (c and e) independent experiments. Error bars denote SEM ***, P
    Figure Legend Snippet: SAC is critical for leukocyte transmigration in vivo. (a) Validation of rat anti–mouse CD99 monoclonal antibody, clone 3F11. Flow cytometry analysis of control (shaded) or anti–mouse CD99 (3F11, thick line) mAb binding to parental or mouse CD99-transfected Ba/F3 cells. (b) The ears of wild-type FVB/n mice (age and sex matched littermates) pretreated with DMSO, anti-CD99 (3F11 mAb), or KH7 (5 µmol/kg) were stimulated with croton oil (1%, 20 µl/ear) or carrier (10% olive oil/90% acetone). After 5 h, mice were sacrificed, their ears were harvested, and immunohistochemical staining was performed using anti-PECAM (ECs), anti-MRP14 (neutrophils), and anti–collagen-IV (basement membrane). 3D confocal images were acquired for each sample. (c) Quantification of results above. Percent of leukocytes extravasated within 50 µm of venules per field of view. (d) Model for quantification of site of arrest. Neutrophils were scored as being in one of six positions: luminal (1), apically arrested (2), arrested partway through the endothelium (3), arrested on the basement membrane (4), migrating through the basement membrane (5), or fully extravasated (6). (e) Quantification of the site of arrest for anti-CD99 and KH7-treated animals. (f) The ears of wild-type FVB/n mice (age- and sex-matched littermates) pretreated with anti-CD99 or rat IgG (control) were stimulated with croton oil. After 3 h, mice received dimaprit (10 mg drug/kg animal) or carrier (H 2 O). Mice were sacrificed 2 h later, their tissue stained, and analyzed as described in panel b. (g) Quantification of results. Percent of leukocytes extravasated within 50 µm of venules per field of view. (h) Additional mice pretreated with anti-CD99 mAb were sacrificed at the time dimaprit was given (3 h) to ensure that the anti-CD99 blockade was present throughout the experiments (5 h total). (i) Quantification of the site of arrest for anti-CD99/carrier and anti-CD99/dimaprit-treated animals. 100–200 cells were analyzed per ear. Total PMN per field of view, vessel length, and vessel diameter were equivalent for all conditions tested (not depicted). Images were acquired with a 40× objective ( n = 1.00). Insets show xz-orthogonal view (where yellow bar dissects the vessel) to demonstrate site of neutrophil arrest. Bars, 25 µm. Two to three mice per condition were used for each experiment. Images are representative of two (f) or three (a and b) independent experiments. Data represent the average value of two (g-i) or three (c and e) independent experiments. Error bars denote SEM ***, P

    Techniques Used: Transmigration Assay, In Vivo, Flow Cytometry, Cytometry, Binding Assay, Transfection, Mouse Assay, Immunohistochemistry, Staining

    Inhibiting sAC or PKA blocks leukocyte transmigration . (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described ( Mamdouh et al., 2009 ). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P
    Figure Legend Snippet: Inhibiting sAC or PKA blocks leukocyte transmigration . (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described ( Mamdouh et al., 2009 ). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P

    Techniques Used: Transmigration Assay, Transmission Electron Microscopy, Staining, Incubation, Blocking Assay

    Raising intracellular cAMP reverses anti-CD99 blockade of transmigration and restores TR of the LBRC. (a and b) Quantitative TEM assays were performed using HUVECs pretreated with anti–VE-cadherin (control) or anti-CD99 mAb (IgG 1 ). After 50 min, 8-CPT (30 µM), Forskolin (30 µM), or DMSO (control) was added to the cells for 10 min. (c) Two-color TR assays were performed (as previously described). In brief, before warming monolayers to 37°C, 488-GαM IgG 2a and 8-CPT, Forskolin, or DMSO were added. Cells were then incubated at 37°C for either 0 or 5 min and subsequently washed, fixed, and stained. Arrows denote LBRC enrichment around anti-CD99–arrested monocytes. (d and e) LBRC enrichment was quantified for both 550- or 488-GαM antibodies. (f) Quantitative TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (control) or anti-CD99 mAb. PBMCs were then added and allowed to transmigrate at 37°C for 50 min. 10 min before fixation, either 8-CPT (general cAMP analogue) or 007-AM (selective-Epac activator) was added to cells. (g) HUVECs were treated in parallel with either 8-CPT or 007-AM for 10 min and then lysed. Immunoblot analysis of pVASP-S157 normalized to total VASP was used to assess PKA activity induced by the drugs. (h) Quantification of results above. Bars, 10 µm. Images are representative of two (c) or three (g) independent experiments. Numerical values are the average of two (d and e) or three (a, b, f, and h) independent experiments. Error bars represent SD (h) or SEM (a, b, and d–f; **, P
    Figure Legend Snippet: Raising intracellular cAMP reverses anti-CD99 blockade of transmigration and restores TR of the LBRC. (a and b) Quantitative TEM assays were performed using HUVECs pretreated with anti–VE-cadherin (control) or anti-CD99 mAb (IgG 1 ). After 50 min, 8-CPT (30 µM), Forskolin (30 µM), or DMSO (control) was added to the cells for 10 min. (c) Two-color TR assays were performed (as previously described). In brief, before warming monolayers to 37°C, 488-GαM IgG 2a and 8-CPT, Forskolin, or DMSO were added. Cells were then incubated at 37°C for either 0 or 5 min and subsequently washed, fixed, and stained. Arrows denote LBRC enrichment around anti-CD99–arrested monocytes. (d and e) LBRC enrichment was quantified for both 550- or 488-GαM antibodies. (f) Quantitative TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (control) or anti-CD99 mAb. PBMCs were then added and allowed to transmigrate at 37°C for 50 min. 10 min before fixation, either 8-CPT (general cAMP analogue) or 007-AM (selective-Epac activator) was added to cells. (g) HUVECs were treated in parallel with either 8-CPT or 007-AM for 10 min and then lysed. Immunoblot analysis of pVASP-S157 normalized to total VASP was used to assess PKA activity induced by the drugs. (h) Quantification of results above. Bars, 10 µm. Images are representative of two (c) or three (g) independent experiments. Numerical values are the average of two (d and e) or three (a, b, f, and h) independent experiments. Error bars represent SD (h) or SEM (a, b, and d–f; **, P

    Techniques Used: Transmigration Assay, Transmission Electron Microscopy, Cycling Probe Technology, Incubation, Staining, Activity Assay

    CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG 1 ) or mouse IgG 1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described ( Mamdouh et al., 2003 ). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG 1 ) or mouse IgG 1 (control). DyLight550 GαM IgG 2a (550-GαM IgG 2a , first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG 2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG 2a (GαM IgG 2a , second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM 2a . (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG 1 , as described above). Before incubation of samples with 488-GαM IgG 2a at 37°C, cells were treated with either GαM IgG 1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P
    Figure Legend Snippet: CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG 1 ) or mouse IgG 1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described ( Mamdouh et al., 2003 ). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG 1 ) or mouse IgG 1 (control). DyLight550 GαM IgG 2a (550-GαM IgG 2a , first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG 2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG 2a (GαM IgG 2a , second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM 2a . (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG 1 , as described above). Before incubation of samples with 488-GαM IgG 2a at 37°C, cells were treated with either GαM IgG 1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P

    Techniques Used: Transmigration Assay, Fluorescence, Staining, Transmission Electron Microscopy, Labeling, Incubation

    5) Product Images from "Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall"

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00872-16

    NbSWP9 contributes to the germination process of N. bombycis . Purified mature N. bombycis spores were induced to germinate in 0.1 mol K 2 CO 3 for 30 min at 28°C. (A and B) DAPI was used to distinguish ungerminated spores (A) and germinated spores (B). The nuclei of ungerminated spores were stained with DAPI as a control. The nuclei of germinated spores are not stained with DAPI. This was evaluated as germinated spores of N. bombycis in this paper. Bar, 10 μm. (C and D) SDS-PAGE and Western blotting were performed to validate whether the supernatant of germinated spores contained NbSWP9. (C) Silver staining was used to detect the supernatant of germinated spores. (D) Western blotting using the anti-NbSWP9 mouse antibody as a primary antibody to detect NbSWP9. (E) Anti-NbSWP9 antibody inhibited spore germination in a dose-dependent manner. In the germination assay, NbSWP9-specific or negative-control antibodies were incubated with N. bombycis spores at different doses (2.5, 5, 7.5, and 10 μg) of IgG that had been purified from sera of rabbits immunized with the recombinant NbSWP9 protein or immunized with PBS (negative-control antibody). Additionally, data are shown as percentages of germinated spores relative to the number of spores of a control sample in which spores were incubated with only 0.1 mol/liter K 2 CO 3 (pH 8.0) at 28°C for 30 min. The percent germination of the control sample was considered to be 100%. “*” represents a significant difference; “**” represents a highly significant difference. The above-described experiments were repeated three times and produced similar results each time. Results from at least 40 random fields were calculated for each data point.
    Figure Legend Snippet: NbSWP9 contributes to the germination process of N. bombycis . Purified mature N. bombycis spores were induced to germinate in 0.1 mol K 2 CO 3 for 30 min at 28°C. (A and B) DAPI was used to distinguish ungerminated spores (A) and germinated spores (B). The nuclei of ungerminated spores were stained with DAPI as a control. The nuclei of germinated spores are not stained with DAPI. This was evaluated as germinated spores of N. bombycis in this paper. Bar, 10 μm. (C and D) SDS-PAGE and Western blotting were performed to validate whether the supernatant of germinated spores contained NbSWP9. (C) Silver staining was used to detect the supernatant of germinated spores. (D) Western blotting using the anti-NbSWP9 mouse antibody as a primary antibody to detect NbSWP9. (E) Anti-NbSWP9 antibody inhibited spore germination in a dose-dependent manner. In the germination assay, NbSWP9-specific or negative-control antibodies were incubated with N. bombycis spores at different doses (2.5, 5, 7.5, and 10 μg) of IgG that had been purified from sera of rabbits immunized with the recombinant NbSWP9 protein or immunized with PBS (negative-control antibody). Additionally, data are shown as percentages of germinated spores relative to the number of spores of a control sample in which spores were incubated with only 0.1 mol/liter K 2 CO 3 (pH 8.0) at 28°C for 30 min. The percent germination of the control sample was considered to be 100%. “*” represents a significant difference; “**” represents a highly significant difference. The above-described experiments were repeated three times and produced similar results each time. Results from at least 40 random fields were calculated for each data point.

    Techniques Used: Purification, Staining, SDS Page, Western Blot, Silver Staining, Germination Assay, Negative Control, Incubation, Recombinant, Produced

    6) Product Images from "The Tetraspanin CD53 Modulates Responses from Activating NK Cell Receptors, Promoting LFA-1 Activation and Dampening NK Cell Effector Functions"

    Article Title: The Tetraspanin CD53 Modulates Responses from Activating NK Cell Receptors, Promoting LFA-1 Activation and Dampening NK Cell Effector Functions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097844

    CD53 induces CD2-independent homotypic clustering of NK cells. A) Purity of 12 day-old LAK cells, as determined by staining with anti-CD3 and anti-NKR-P1A. LAK cells were cultured for 2 h in the presence of soluble antibodies towards CD53, CD2, NKR-P1A, or an isotype control IgG. B) Cluster formation was photographed with a light microscope, or C) quantified by counting the number of free, non-aggregated cells in treated samples relative to cells cultured in medium using a hemocytometer. D and E) LAK cells were co-cultured for up to 2 h with antibodies towards CD53 and CD2 or with CD53 and isotype control antibody. Cluster formation was assessed qualitatively by microscopy (D), or quantitatively by hemocytometer (E) as described above. Results are presented as the mean±SEM of 3 independent experiments with samples in duplicates (n = 6). Comparisons within an experimental group were performed with the One-way analysis of variance (ANOVA) and a post hoc Tukey's multiple comparisons test to compare treated samples to untreated sample. *, p
    Figure Legend Snippet: CD53 induces CD2-independent homotypic clustering of NK cells. A) Purity of 12 day-old LAK cells, as determined by staining with anti-CD3 and anti-NKR-P1A. LAK cells were cultured for 2 h in the presence of soluble antibodies towards CD53, CD2, NKR-P1A, or an isotype control IgG. B) Cluster formation was photographed with a light microscope, or C) quantified by counting the number of free, non-aggregated cells in treated samples relative to cells cultured in medium using a hemocytometer. D and E) LAK cells were co-cultured for up to 2 h with antibodies towards CD53 and CD2 or with CD53 and isotype control antibody. Cluster formation was assessed qualitatively by microscopy (D), or quantitatively by hemocytometer (E) as described above. Results are presented as the mean±SEM of 3 independent experiments with samples in duplicates (n = 6). Comparisons within an experimental group were performed with the One-way analysis of variance (ANOVA) and a post hoc Tukey's multiple comparisons test to compare treated samples to untreated sample. *, p

    Techniques Used: Staining, Cell Culture, Light Microscopy, Microscopy

    7) Product Images from "TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development"

    Article Title: TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145277

    CC and MIT domains are required for stable association of TMF with the Golgi. (A) Diagram of the TMF aa sequence with the marked MIT and CC motifs (top). Constructs 1–5 transfected into NIH3T3 cells are depicted below. The TMF aa included in each construct are shown, and the presence of the CC or MIT motifs in each construct are marked in the brackets. (B-F) Confocal microscopy analysis of EGFP-fused TMF segments, ectopically expressed (green) in NIH3T3 cells. (G) Confocal microscopy analysis of TMF segment containing the MIT and c-terminus CC domain fused to EGFP (EGFP-TMF C5) (green) overlapping the immuno-staining of GM130 (Golgi marker, red.). Arrows represent co-localization (yellow color). The different fluorescence channels are presented in the right panels. Bar represents 20μm. Nuclei were visualized with Hoechst solution (blue). (H) As in G , only that these cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (I) Confocal microscope analysis of the full TMF protein fused to EGFP (green) overlapping the immuno-stained Golgi (golgin 97- red.) (J) As in I , only that cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Nuclei were visualized with Hoechst solution (blue).Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (K) IP of GFP-TMF protein fragments from lysates of NIH3T3 cells expressing either GFP-TMF containing the MIT domain (TMF-MIT) or GFP-TMF fragment containing the CC domains without the MIT motif (TMF-CC).Whole cell lysate (WCL) represents the input of the IP. IgG beads served as a negative control. The IP was followed by a WB analysis to assess the co-IP of the TMF fragment and tubulin. Images represent one out of five independent typical WB analyses which gave similar results.
    Figure Legend Snippet: CC and MIT domains are required for stable association of TMF with the Golgi. (A) Diagram of the TMF aa sequence with the marked MIT and CC motifs (top). Constructs 1–5 transfected into NIH3T3 cells are depicted below. The TMF aa included in each construct are shown, and the presence of the CC or MIT motifs in each construct are marked in the brackets. (B-F) Confocal microscopy analysis of EGFP-fused TMF segments, ectopically expressed (green) in NIH3T3 cells. (G) Confocal microscopy analysis of TMF segment containing the MIT and c-terminus CC domain fused to EGFP (EGFP-TMF C5) (green) overlapping the immuno-staining of GM130 (Golgi marker, red.). Arrows represent co-localization (yellow color). The different fluorescence channels are presented in the right panels. Bar represents 20μm. Nuclei were visualized with Hoechst solution (blue). (H) As in G , only that these cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (I) Confocal microscope analysis of the full TMF protein fused to EGFP (green) overlapping the immuno-stained Golgi (golgin 97- red.) (J) As in I , only that cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Nuclei were visualized with Hoechst solution (blue).Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (K) IP of GFP-TMF protein fragments from lysates of NIH3T3 cells expressing either GFP-TMF containing the MIT domain (TMF-MIT) or GFP-TMF fragment containing the CC domains without the MIT motif (TMF-CC).Whole cell lysate (WCL) represents the input of the IP. IgG beads served as a negative control. The IP was followed by a WB analysis to assess the co-IP of the TMF fragment and tubulin. Images represent one out of five independent typical WB analyses which gave similar results.

    Techniques Used: Sequencing, Construct, Transfection, Confocal Microscopy, Immunostaining, Marker, Fluorescence, Staining, Microscopy, Expressing, Negative Control, Western Blot, Co-Immunoprecipitation Assay

    8) Product Images from "A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus"

    Article Title: A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136888

    BALB/c mice immunized with MAP27 produced anti-MAP27, anti-PGN and anti- S . aureus antibodies. (A) Titers of anti-MAP27 serum during the period of immunization. 96-well plates were coated with MAP27. Serum samples from mice immunized with MAP27, MAPctrl or blank were pooled and added in a 10-fold serial dilution, followed by incubation with HRP-conjunctive goat anti-mouse IgG. (B) Sera from MAP27-immunized mice bind to PGN after the fifth MAP immunization. (C) Sera from MAP27-immunized mice bind to S . aureus after boosting with heat-killed S . aureus . For ELISA assays in panel B and C, 96-well plates were coated with PGN or sonicated S . aureus fragments. Serum samples were added in a 1/200 dilution as primary antibodies, followed by incubation with HRP-conjunctive goat anti-mouse IgG. The absorbance was measured at OD 450nm . The results are presented as means ±SEM. * P
    Figure Legend Snippet: BALB/c mice immunized with MAP27 produced anti-MAP27, anti-PGN and anti- S . aureus antibodies. (A) Titers of anti-MAP27 serum during the period of immunization. 96-well plates were coated with MAP27. Serum samples from mice immunized with MAP27, MAPctrl or blank were pooled and added in a 10-fold serial dilution, followed by incubation with HRP-conjunctive goat anti-mouse IgG. (B) Sera from MAP27-immunized mice bind to PGN after the fifth MAP immunization. (C) Sera from MAP27-immunized mice bind to S . aureus after boosting with heat-killed S . aureus . For ELISA assays in panel B and C, 96-well plates were coated with PGN or sonicated S . aureus fragments. Serum samples were added in a 1/200 dilution as primary antibodies, followed by incubation with HRP-conjunctive goat anti-mouse IgG. The absorbance was measured at OD 450nm . The results are presented as means ±SEM. * P

    Techniques Used: Mouse Assay, Produced, Serial Dilution, Incubation, Enzyme-linked Immunosorbent Assay, Sonication

    9) Product Images from "Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura"

    Article Title: Generation of Anti-Murine ADAMTS13 Antibodies and Their Application in a Mouse Model for Acquired Thrombotic Thrombocytopenic Purpura

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0160388

    Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.
    Figure Legend Snippet: Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7. Adamts13 +/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions ( S1 Fig ). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [ > 10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.

    Techniques Used: Ex Vivo, Mouse Assay, Injection, Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Molecular Weight, Software

    Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.
    Figure Legend Snippet: Development of a sensitive mADAMTS13 detection assay. (A) Binding of plasma mADAMTS13 to the anti-mADAMTS13 mAbs was tested in ELISA. The respective anti-mMDTCS (black) and anti-mT2-CUB2 (white) mAbs were coated on a 96-well microtiter plate. After blocking, plasma mADAMTS13 was added (0.1 U/mL) and was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (B) The same ELISA was performed as in A in triplicate for anti-mT2-CUB2 mAb 9F2, anti-mMDTCS mAb 14H7 and the previously reported anti-mMDTCS mAb 20A10 [ 31 ]. Binding was calculated relative to binding of 0.10 U/mL plasma mADAMTS13 to 20A10 (which was set to a value of ‘1.0’). Error bars represent the SD of the three independently performed experiments.

    Techniques Used: Detection Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

    Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.
    Figure Legend Snippet: Epitope mapping and epitope overview of the developed anti-mADAMTS13 mAbs. The epitope of each anti-mADAMTS13 mAb was mapped against both mMDTCS (A) and mT2-CUB2 (B). Individual anti-mADAMTS13 mAbs were coated, recombinant mMDTCS (A) or mT2-CUB2 (B) were added and binding of the respective mADAMTS13 variant was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. Black and white bars represent respectively anti-mMDTCS and anti-mT2-CUB2 mAbs. The previously reported mAb 20A10 [ 31 ] was used as a positive (A) and negative (B) control. (C) Epitope overview of the developed anti-mADAMTS13 mAbs. The previously developed mAb 20A10 [ 31 ] is marked by a dark frame.

    Techniques Used: Recombinant, Binding Assay, Variant Assay

    10) Product Images from "Podocalyxin in Rat Platelets and Megakaryocytes"

    Article Title: Podocalyxin in Rat Platelets and Megakaryocytes

    Journal: The American Journal of Pathology

    doi:

    Podocalyxin ( a ) and CD62P antigens ( b ) in a megakaryocyte, shown by double IF staining of a cytospin slide of rat bone marrow cells first with monoclonal anti-CD62P and TRITC-conjugated goat anti-mouse IgG ( b ), followed by FITC-conjugated 5A IgG ( a ). Binding to the anti-mouse conjugate was blocked by pretreatment with normal mouse serum. The polyploid megakaryocyte expresses both antigens. Anti-podocalyxin antibodies detect also a small, unidentified cell ( a , arrow ). Magnification, ×600.
    Figure Legend Snippet: Podocalyxin ( a ) and CD62P antigens ( b ) in a megakaryocyte, shown by double IF staining of a cytospin slide of rat bone marrow cells first with monoclonal anti-CD62P and TRITC-conjugated goat anti-mouse IgG ( b ), followed by FITC-conjugated 5A IgG ( a ). Binding to the anti-mouse conjugate was blocked by pretreatment with normal mouse serum. The polyploid megakaryocyte expresses both antigens. Anti-podocalyxin antibodies detect also a small, unidentified cell ( a , arrow ). Magnification, ×600.

    Techniques Used: Staining, Binding Assay

    11) Product Images from "“On silico” peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions"

    Article Title: “On silico” peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions

    Journal: Nature medicine

    doi: 10.1038/nm.2913

    Epitope mapping of polyclonal and monoclonal antibodies using H2B Intel arrays ( a ) Single-amino acid resolution map of the epitope recognized by H2B-reactive polyclonal antibody (pAb) 18977 (Abcam, Cambridge, MA) showing that a minimum of two N-terminal amino acids (Pro1–Glu2, inset) are required for antibody binding. ( b ) Epitope map for monoclonal antibody (mAb) 52988 (Abcam) directed against residues surrounding Lys20-Ac, showing a minimum binding requirement of either Ala17–Lys20-Ac or Val18–Ala21 (containing Lys20-Ac). ( c ) Epitope map and minimum peptide binding requirements for mAb 62335 (Abcam) directed against residues surrounding Lys15-Ac of H2B. The minimum binding requirements were determined to be Gly13–Lys15-Ac and Lys15-Ac–Lys16. ( d ) Epitope map for mAb 40886 (Abcam) directed against residues surrounding Lys5-Ac, showing binding of acetylated lysine (Lys-Ac), both at position 5 in the context of at least a 3-mer peptide Ala4-Ser6, but also Lys-Ac alone or in peptides at positions 12, 15 and 20. For each antibody, the “All (Ac)” subarray containing acetylated lysine at position 5, 12, 15 and 20 is enlarged. Grid schematic for each antibody corresponds to reactivity within the enlarged “All (Ac)” subarray. All primary antibodies were diluted 1:1000 from original stocks as received from vendors and reactive features were visualized with goat anti-rabbit IgG conjugated to Cy5. Color bars indicate observed median fluorescence intensity (MFI) at each peptide feature.
    Figure Legend Snippet: Epitope mapping of polyclonal and monoclonal antibodies using H2B Intel arrays ( a ) Single-amino acid resolution map of the epitope recognized by H2B-reactive polyclonal antibody (pAb) 18977 (Abcam, Cambridge, MA) showing that a minimum of two N-terminal amino acids (Pro1–Glu2, inset) are required for antibody binding. ( b ) Epitope map for monoclonal antibody (mAb) 52988 (Abcam) directed against residues surrounding Lys20-Ac, showing a minimum binding requirement of either Ala17–Lys20-Ac or Val18–Ala21 (containing Lys20-Ac). ( c ) Epitope map and minimum peptide binding requirements for mAb 62335 (Abcam) directed against residues surrounding Lys15-Ac of H2B. The minimum binding requirements were determined to be Gly13–Lys15-Ac and Lys15-Ac–Lys16. ( d ) Epitope map for mAb 40886 (Abcam) directed against residues surrounding Lys5-Ac, showing binding of acetylated lysine (Lys-Ac), both at position 5 in the context of at least a 3-mer peptide Ala4-Ser6, but also Lys-Ac alone or in peptides at positions 12, 15 and 20. For each antibody, the “All (Ac)” subarray containing acetylated lysine at position 5, 12, 15 and 20 is enlarged. Grid schematic for each antibody corresponds to reactivity within the enlarged “All (Ac)” subarray. All primary antibodies were diluted 1:1000 from original stocks as received from vendors and reactive features were visualized with goat anti-rabbit IgG conjugated to Cy5. Color bars indicate observed median fluorescence intensity (MFI) at each peptide feature.

    Techniques Used: Binding Assay, Fluorescence

    12) Product Images from "Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes"

    Article Title: Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2( SK2) in mouse cardiac myocytes, et al. Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

    Journal: Acta Physiologica (Oxford, England)

    doi: 10.1111/apha.12986

    Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm
    Figure Legend Snippet: Colocalizations of JP 2 and SK 2 channels in adult mouse cardiac myocytes and HEK 293 cells. Confocal images show double immunostaining with anti‐ JP 2 antibody (green) and anti‐ SK 2 antibody (red) in single isolated mouse atria (A) and ventricular (B) myocytes as well as HEK 293 cells (D) cotransfected with JP 2 and SK 2. Negative control experiments (Control) were performed with the secondary antibodies with anti‐rabbit‐IgG TRITC ‐conjugated and with anti‐mouse‐IgG FITC ‐conjugated in atria myocytes (C) and HEK 293 cells (E). Merge images show the colocalization of JP 2 and SK 2 channels near the Z ‐lines in the cardiac cells or on the surface membrane of HEK 293 cells. Scale bars, 10 μm

    Techniques Used: Double Immunostaining, Isolation, Negative Control

    13) Product Images from "Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments"

    Article Title: Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200401078

    Inhibition of the endocytosis of E-cadherin by trans-interacting E-cadherin or trans-interacting nectin-1 in intact cells. (A) Inhibition of the constitutive endocytosis of E-cadherin by trans-interacting E-cadherin in intact EL cells. (Aa) EL cells were incubated in the medium with 400 nM Cef or 400 nM human IgG (control IgG) for 60 min. The cells were surface-biotinylated on ice and cultured at 18°C for the indicated periods of time to allow the endocytosis of E-cadherin. Biotinylated proteins on the plasma membrane were then stripped off by glutathione treatment, and biotinylated proteins inside the cells were recovered on streptavidin beads. The bound proteins were analyzed by immunoblotting with the anti-E-cadherin mAb. The relative amounts of endocytosed E-cadherin were expressed as percentage of total biotinylated E-cadherin in the bottom panel. The mean ± SD of duplicate assays is shown. (Ab) EL cells pretreated with the 400 nM Cef or 400 nM control IgG were incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. EL cells overexpressing GFP, GFP-N17Rac1, or GFP-N-WASP-CRIB-CAAX were pretreated with the 400 nM Cef or 400 nM control IgG and then incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. The cells were then fixed and immunostained with the anti–E-cadherin mAb and the anti-IQGAP1 pAb. Bars, 10 μm. (B) Inhibition of the constitutive endocytosis of E-cadherin by trans-interacting nectin-1 in intact nectin-1-EL cells. (Ba) Nectin-1-EL (N1-EL) cells were incubated in the medium with 400 nM Nef-3 or 400 nM control IgG for 60 min. The cells were surface-biotinylated on ice and cultured at 18°C for the indicated time to allow the endocytosis of E-cadherin. Biotinylated proteins on the plasma membranes were then stripped off by glutathione treatment, and biotinylated proteins inside the cells were recovered on streptavidin beads. The bound proteins were analyzed by immunoblotting with the anti–E-cadherin mAb. The relative amounts of endocytosed E-cadherin were expressed as percentage of total biotinylated E-cadherin in the bottom panel. The mean ± SD of duplicate assays is shown. (Bb) N1-EL cells pretreated with 400 nM Nef-3 or 400 nM control IgG were incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. N1-EL cells overexpressing GFP, GFP-N17Rac1, or GFP-N-WASP-CRIB-CAAX were pretreated with the 400 nM Nef-3 or 400 nM control IgG and then incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. The cells were then fixed and immunostained with the anti–E-cadherin mAb and the anti-IQGAP1 pAb. Bars, 10 μm. The results shown in all panels are representative of at least three independent experiments.
    Figure Legend Snippet: Inhibition of the endocytosis of E-cadherin by trans-interacting E-cadherin or trans-interacting nectin-1 in intact cells. (A) Inhibition of the constitutive endocytosis of E-cadherin by trans-interacting E-cadherin in intact EL cells. (Aa) EL cells were incubated in the medium with 400 nM Cef or 400 nM human IgG (control IgG) for 60 min. The cells were surface-biotinylated on ice and cultured at 18°C for the indicated periods of time to allow the endocytosis of E-cadherin. Biotinylated proteins on the plasma membrane were then stripped off by glutathione treatment, and biotinylated proteins inside the cells were recovered on streptavidin beads. The bound proteins were analyzed by immunoblotting with the anti-E-cadherin mAb. The relative amounts of endocytosed E-cadherin were expressed as percentage of total biotinylated E-cadherin in the bottom panel. The mean ± SD of duplicate assays is shown. (Ab) EL cells pretreated with the 400 nM Cef or 400 nM control IgG were incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. EL cells overexpressing GFP, GFP-N17Rac1, or GFP-N-WASP-CRIB-CAAX were pretreated with the 400 nM Cef or 400 nM control IgG and then incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. The cells were then fixed and immunostained with the anti–E-cadherin mAb and the anti-IQGAP1 pAb. Bars, 10 μm. (B) Inhibition of the constitutive endocytosis of E-cadherin by trans-interacting nectin-1 in intact nectin-1-EL cells. (Ba) Nectin-1-EL (N1-EL) cells were incubated in the medium with 400 nM Nef-3 or 400 nM control IgG for 60 min. The cells were surface-biotinylated on ice and cultured at 18°C for the indicated time to allow the endocytosis of E-cadherin. Biotinylated proteins on the plasma membranes were then stripped off by glutathione treatment, and biotinylated proteins inside the cells were recovered on streptavidin beads. The bound proteins were analyzed by immunoblotting with the anti–E-cadherin mAb. The relative amounts of endocytosed E-cadherin were expressed as percentage of total biotinylated E-cadherin in the bottom panel. The mean ± SD of duplicate assays is shown. (Bb) N1-EL cells pretreated with 400 nM Nef-3 or 400 nM control IgG were incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. N1-EL cells overexpressing GFP, GFP-N17Rac1, or GFP-N-WASP-CRIB-CAAX were pretreated with the 400 nM Nef-3 or 400 nM control IgG and then incubated at 18°C for 20 min to allow the endocytosis of E-cadherin. The cells were then fixed and immunostained with the anti–E-cadherin mAb and the anti-IQGAP1 pAb. Bars, 10 μm. The results shown in all panels are representative of at least three independent experiments.

    Techniques Used: Inhibition, Incubation, Cell Culture

    Characteristics of the endocytosed vesicles of E-cadherin. (A) Specific endocytosis of E-cadherin. The relative amount of the MSP and the HSP (15 μg of protein in each fraction) were analyzed by quantitative immunoblotting with various antibodies against the indicated proteins. The results were obtained from the same experiments and the same gels. (B) Isolation of endocytosed vesicles of E-cadherin. (Ba) Electron microscopic morphology of the HSP. The reaction for the formation of vesicles was performed as shown in Fig. 1 A. Bar, 100 nm. (Bb) Immunoelectron microscopic morphology of endocytosed vesicles of E-cadherin. The reaction for the formation of vesicles was performed as shown in Fig. 1 A. The vesicles were isolated from the MSS using the anti– β-catenin mAb-coated magnetic beads. The accumulated vesicles on the beads were processed for electron microscopy. The membranes bound to the beads consisted largely of a homogenous population of 60–80-nm vesicles (arrowheads). (inset) Immunogold labeling for E-cadherin. The immunoisolated beads were stained with the anti-pan-cadherin (cytoplasmic portion) pAb, followed by 10 nm gold-conjugated secondary antibody (arrows). Bars, 100 nm. (Bc) Composition of the endocytosed vesicles of E-cadherin. The vesicles were immunoisolated with the anti–β-catenin mAb or the anti-mouse IgG (control IgG)-coated magnetic beads from the MSS as shown in Bb. The bound proteins were analyzed by immunoblotting with various antibodies against the indicated proteins. The results were obtained from the same experiments and the same gels. The results shown in all panels are representative of three independent experiments.
    Figure Legend Snippet: Characteristics of the endocytosed vesicles of E-cadherin. (A) Specific endocytosis of E-cadherin. The relative amount of the MSP and the HSP (15 μg of protein in each fraction) were analyzed by quantitative immunoblotting with various antibodies against the indicated proteins. The results were obtained from the same experiments and the same gels. (B) Isolation of endocytosed vesicles of E-cadherin. (Ba) Electron microscopic morphology of the HSP. The reaction for the formation of vesicles was performed as shown in Fig. 1 A. Bar, 100 nm. (Bb) Immunoelectron microscopic morphology of endocytosed vesicles of E-cadherin. The reaction for the formation of vesicles was performed as shown in Fig. 1 A. The vesicles were isolated from the MSS using the anti– β-catenin mAb-coated magnetic beads. The accumulated vesicles on the beads were processed for electron microscopy. The membranes bound to the beads consisted largely of a homogenous population of 60–80-nm vesicles (arrowheads). (inset) Immunogold labeling for E-cadherin. The immunoisolated beads were stained with the anti-pan-cadherin (cytoplasmic portion) pAb, followed by 10 nm gold-conjugated secondary antibody (arrows). Bars, 100 nm. (Bc) Composition of the endocytosed vesicles of E-cadherin. The vesicles were immunoisolated with the anti–β-catenin mAb or the anti-mouse IgG (control IgG)-coated magnetic beads from the MSS as shown in Bb. The bound proteins were analyzed by immunoblotting with various antibodies against the indicated proteins. The results were obtained from the same experiments and the same gels. The results shown in all panels are representative of three independent experiments.

    Techniques Used: Isolation, Magnetic Beads, Electron Microscopy, Labeling, Staining

    14) Product Images from "Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter"

    Article Title: Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001292

    PfH2A.Z is expressed in the nucleus throughout asexual differentiation. Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1 st panel). Pre-immune serum (pI) does not show any reactivity (2 nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3 rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4 th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R = ring stage, T = trophozoite stage, S = schizont stage. DIC = differential interference contrast.
    Figure Legend Snippet: PfH2A.Z is expressed in the nucleus throughout asexual differentiation. Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1 st panel). Pre-immune serum (pI) does not show any reactivity (2 nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3 rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4 th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R = ring stage, T = trophozoite stage, S = schizont stage. DIC = differential interference contrast.

    Techniques Used: SDS Page, Western Blot, Immunoprecipitation, Positive Control, Expressing, Immunofluorescence, Confocal Microscopy

    15) Product Images from "Small, Membrane-bound, Alternatively Spliced Forms of Ankyrin 1 Associated with the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle"

    Article Title: Small, Membrane-bound, Alternatively Spliced Forms of Ankyrin 1 Associated with the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle

    Journal: The Journal of Cell Biology

    doi:

    Comparison of the distribution of the small ankyrins and the SERCA1 ATPase in rat skeletal muscle. Frozen cross sections (5 μm) of the extensor digitorum longus muscle of the rat were prepared and double-labeled by immunofluorescence with anti-p6 antibodies to the small ankyrins and monoclonal antibodies to the SERCA1 ATPase of fast twitch muscle fibers, as described in Materials and Methods. Images were obtained by confocal microscopy. ( A ) SERCA ATPase, visualized with fluoresceinated anti-mouse IgG. ( B ) Small ankyrins, visualized with tetramethylrhodamine-conjugated anti-rabbit IgG. ( C ) Computer overlay of the images in A and B , in which structures labeled by both antibodies appear yellow. There is extensive coincidence of the two labels. Bar, 6 μm.
    Figure Legend Snippet: Comparison of the distribution of the small ankyrins and the SERCA1 ATPase in rat skeletal muscle. Frozen cross sections (5 μm) of the extensor digitorum longus muscle of the rat were prepared and double-labeled by immunofluorescence with anti-p6 antibodies to the small ankyrins and monoclonal antibodies to the SERCA1 ATPase of fast twitch muscle fibers, as described in Materials and Methods. Images were obtained by confocal microscopy. ( A ) SERCA ATPase, visualized with fluoresceinated anti-mouse IgG. ( B ) Small ankyrins, visualized with tetramethylrhodamine-conjugated anti-rabbit IgG. ( C ) Computer overlay of the images in A and B , in which structures labeled by both antibodies appear yellow. There is extensive coincidence of the two labels. Bar, 6 μm.

    Techniques Used: Labeling, Immunofluorescence, Confocal Microscopy

    16) Product Images from "Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis"

    Article Title: Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20050433

    (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.
    Figure Legend Snippet: (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.

    Techniques Used: Staining, Incubation, Microscopy, Translocation Assay, Fractionation, Western Blot, Marker

    (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.
    Figure Legend Snippet: (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.

    Techniques Used: Incubation, Flow Cytometry, Cytometry, Activation Assay, Staining, Expressing, Fluorescence

    17) Product Images from "Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation"

    Article Title: Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation

    Journal: JCI Insight

    doi: 10.1172/jci.insight.127395

    CaMK4 binds to PKM2 and increases pyruvate kinase activity. ( A and B ) HEK293T cells were transfected with Flag-hCaMK4 and 6×His-hPKM2 or 6×His-tagged enhanced green fluorescent protein (His-EGFP), followed by coimmunoprecipitation using anti-Flag, anti-6×His, or anti–mouse IgG Abs. Western blotting was performed using anti-Flag ( A ) or 6×His ( B ) Abs. Data are representative of 3 independent experiments. ( C ) Camk4 -deficient naive CD4 + T cells were transfected with Flag-mCaMK4 under Th17-polarizing conditions, followed by coimmunoprecipitation using Flag or mouse IgG Abs. Western blotting was performed using PKM2 Ab. Data are representative of 2 independent experiments. ( D ) Camk4 -sufficient or -deficient naive CD4 + T cells were cultured with CD3 and CD28 Abs for 48 hours in vitro and after 2-hour pretreatment with ionomycin, cells were collected and pyruvate kinase activity was measured. Cumulative data are shown (mean ± SEM); n = 3. ** P
    Figure Legend Snippet: CaMK4 binds to PKM2 and increases pyruvate kinase activity. ( A and B ) HEK293T cells were transfected with Flag-hCaMK4 and 6×His-hPKM2 or 6×His-tagged enhanced green fluorescent protein (His-EGFP), followed by coimmunoprecipitation using anti-Flag, anti-6×His, or anti–mouse IgG Abs. Western blotting was performed using anti-Flag ( A ) or 6×His ( B ) Abs. Data are representative of 3 independent experiments. ( C ) Camk4 -deficient naive CD4 + T cells were transfected with Flag-mCaMK4 under Th17-polarizing conditions, followed by coimmunoprecipitation using Flag or mouse IgG Abs. Western blotting was performed using PKM2 Ab. Data are representative of 2 independent experiments. ( D ) Camk4 -sufficient or -deficient naive CD4 + T cells were cultured with CD3 and CD28 Abs for 48 hours in vitro and after 2-hour pretreatment with ionomycin, cells were collected and pyruvate kinase activity was measured. Cumulative data are shown (mean ± SEM); n = 3. ** P

    Techniques Used: Activity Assay, Transfection, Western Blot, Cell Culture, In Vitro

    18) Product Images from "Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo"

    Article Title: Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.8.4819-4827.2005

    Localization of Flag-tagged ORF35 protein in infected melanoma cells. Melanoma cells infected with rOkaΔ35/35Flag@Avr were fixed and permeabilized at 24 and 96 h after inoculation. Infected cells were incubated with primary polyclonal rabbit antibody to the Flag tag, fluorescein isothiocyanate-conjugated anti-rabbit IgG (green), monoclonal anti-VZV gE antibody, Texas red-conjugated anti-mouse IgG (red) antibody, and the nuclear marker, DAPI (blue).
    Figure Legend Snippet: Localization of Flag-tagged ORF35 protein in infected melanoma cells. Melanoma cells infected with rOkaΔ35/35Flag@Avr were fixed and permeabilized at 24 and 96 h after inoculation. Infected cells were incubated with primary polyclonal rabbit antibody to the Flag tag, fluorescein isothiocyanate-conjugated anti-rabbit IgG (green), monoclonal anti-VZV gE antibody, Texas red-conjugated anti-mouse IgG (red) antibody, and the nuclear marker, DAPI (blue).

    Techniques Used: Infection, Incubation, FLAG-tag, Marker

    19) Product Images from "Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice"

    Article Title: Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice

    Journal: Journal of Clinical Investigation

    doi:

    Detection of iNOS in livers of Con A–treated mice by immunofluorescence staining. ( a ) Con A–induced iNOS expression in livers of BALB/c mice was detected by immunofluorescence staining of liver cryostat sections (rabbit anti-mouse iNOS antiserum, secondary goat anti-rabbit IgG tagged with Cy3; red fluorescence). ( b ) Costaining of iNOS (rabbit anti-mouse iNOS antiserum, secondary swine anti-rabbit IgG tagged with FITC; green fluorescence) and Kupffer cells (BM8, secondary goat anti-rat IgG tagged with Texas red; red fluorescence) 4 hours after Con A or GalN/LPS treatment. All sections were examined by confocal laser-scanning microscopy. Costaining is represented by yellow fluorescence. Seen in a and b is one example of three independent experiments, respectively.
    Figure Legend Snippet: Detection of iNOS in livers of Con A–treated mice by immunofluorescence staining. ( a ) Con A–induced iNOS expression in livers of BALB/c mice was detected by immunofluorescence staining of liver cryostat sections (rabbit anti-mouse iNOS antiserum, secondary goat anti-rabbit IgG tagged with Cy3; red fluorescence). ( b ) Costaining of iNOS (rabbit anti-mouse iNOS antiserum, secondary swine anti-rabbit IgG tagged with FITC; green fluorescence) and Kupffer cells (BM8, secondary goat anti-rat IgG tagged with Texas red; red fluorescence) 4 hours after Con A or GalN/LPS treatment. All sections were examined by confocal laser-scanning microscopy. Costaining is represented by yellow fluorescence. Seen in a and b is one example of three independent experiments, respectively.

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, Expressing, Fluorescence, Confocal Laser Scanning Microscopy

    Related Articles

    Activation Assay:

    Article Title: Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis
    Article Snippet: .. Caspase 3 activation was analyzed by incubating rehydrated methanol-fixed cells with antibody to cleaved caspase 3 (Asp 175; Cell Signaling Technologies) followed by FITC-conjugated AffiniPure F(ab)2 -fragment of goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories). .. Subcellular fractionation and Western blot.

    Incubation:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    Blocking Assay:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    Negative Control:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    Staining:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    Transmission Assay:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    Microscopy:

    Article Title: Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall
    Article Snippet: .. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV. .. Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis .

    SDS Page:

    Article Title: The Tetraspanin CD53 Modulates Responses from Activating NK Cell Receptors, Promoting LFA-1 Activation and Dampening NK Cell Effector Functions
    Article Snippet: Cells were immediately lysed by addition of an equal volume of 2x ice-cold lysis buffer (2% Nonidet P-40, 20 mM Na3 VO4 , 2x protease inhibitor cocktail, 2 mM PMSF (all from Sigma), and 2x PhosSTOP phosphatase inhibitor (Roche), 300 mM NaCl and 50 mM Tris, pH 7.4), then spun at 10 K g for 10 min. For immunoprecipitations, Protein G Dynabeads (Dynal, Invitrogen) were pre-coated with relevant antibodies for 30 min at room temperature, prior to incubation with cell lysates for 2 h at 4°C, washed 3x in 1x lysis buffer, and resuspended in SDS sample buffer. .. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore), and detected with primary antibodies in either 3% BSA or 5% skimmed milk in TBS with 0.05% Tween overnight at 4°C, and for 1 h with secondary horseradish peroxidase-conjugated goat anti-mouse IgG, goat-anti mouse IgG2b, or goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). .. Proteins were visualized by enhanced chemiluminescence (Pierce, UK).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Jackson Immuno fluorescein fitc affinipure fab fragment goat anti rabbit igg
    Inactivation of the MLL complex prevents the recruitment of MCM2-7 proteins onto chromatin. (A) HCT116 cells were transfected with 50 nM siRNAs of luciferase, Geminin, Geminin+RBBP5, CDT2, CDT2+RBBP5 and RBBP5. The cells were fixed and stained with anti-MCM7 and <t>FITC-conjugated</t> secondary antibodies and counter-stained with DAPI. Scale bars: 50 μm. (B) Proteins from the siRNA-treated cell lysates in A were analyzed by specific antibodies as indicated. (C) Downregulation of WDR5 or RBBP5 reduces the recruitment of MCM proteins to chromatin. Chromatin fractions were isolated from control and WDR5 or RBBP5 siRNA-treated cells and the chromatin-associated MCM proteins were examined by western blotting as indicated. (D) Relative protein levels of MCMs and methylated histone H3K4 on chromatin in C were quantified using Gel-Pro analyzer 4.0. The error bars indicate standard deviation of triplicated samples. The statistical differences of MCM2, MCM3, MCM7, CDT1 and histone H3K4 methylations between control and specific siRNA samples were analyzed using the two-tailed Student's t -test. (E,F) The chromatin immunoprecipitation (ChIP) analysis was performed to locate MCM7 (E) and MCM2 (F) proteins on the DNA replication origin at the Mcm4 gene. Proteins were cross-linked to chromatin and chromatin DNA was sonicated to generate 500-1000 base-pairs (bps) fragments in average length. The ChIP-grade anti-MCM7 and MCM2 antibodies were used for chromatin immunoprecipitation. Cross-linked DNA was released, purified, and analyzed for the enrichment of DNA fragments associated with MCM2 and MCM7 from −4.0 kb to 4.0 kb (kilobase pairs) along the Mcm4 region using various Mcm4 primers and quantitative real time PCR as described in the Materials and Methods. Error bars indicate the standard deviation of triplicated samples. The statistical significance of antibody-enriched specific Mcm4 DNA sequences over the background control <t>IgG</t> binding (fold enrichment) was assessed using the two-tailed Student's t -test.
    Fluorescein Fitc Affinipure Fab Fragment Goat Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein fitc affinipure fab fragment goat anti rabbit igg/product/Jackson Immuno
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorescein fitc affinipure fab fragment goat anti rabbit igg - by Bioz Stars, 2021-06
    88/100 stars
      Buy from Supplier

    93
    Jackson Immuno alexa 488 conjugated anti rabbit igg secondary antibodies
    Secretion of Type I collagen was induced by purified <t>IgG</t> from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P
    Alexa 488 Conjugated Anti Rabbit Igg Secondary Antibodies, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 488 conjugated anti rabbit igg secondary antibodies/product/Jackson Immuno
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa 488 conjugated anti rabbit igg secondary antibodies - by Bioz Stars, 2021-06
    93/100 stars
      Buy from Supplier

    97
    Jackson Immuno goat anti rabbit igg
    (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and <t>FITC-conjugated</t> goat anti–rabbit <t>IgG,</t> and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.
    Goat Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit igg/product/Jackson Immuno
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg - by Bioz Stars, 2021-06
    97/100 stars
      Buy from Supplier

    93
    Jackson Immuno goat anti rabbit cy3
    A nontoxic dose of ATL induces oxidative DNA damage and activates PARP in cancer cells. The PC-3 prostate cancer and NCM460 normal colon epithelial cells, with or without 1 h of preincubation in 10 mM NAC, were treated by 10 μM ATL or vehicle control for the indicated times. a , b Measurement of ROS in PC-3 cells by flow cytometry. Data from three independent experiments were presented as mean ± SD. c , d Measurement of ROS in NCM460 cells by flow cytometry. e , f Immunofluorescent staining of cellular 8-oxoG by <t>Cy3-conjugated</t> avidin. PC-3 cells were treated by 10 μM ATL for 12 h (scale bar: 10 μm). Nuclear 8-oxoG intensity was measured using the ImageJ software and the data were processed by the Prism software. g Representative images of alkaline comet assay. PC-3 cells were treated by vehicle control or 10 μM ATL for 12 h. h The tail moment was defined as percentage of tail DNA × tail length, quantified using the TriTek CometScore software. i Immunofluorescence staining for PAR foci in PC-3 cells treated by 10 μM ATL for 12 h. DNA was counterstained with DAPI (scale bar: 5 μm). j Western blot analysis of PAR in PC-3 cells. Ten micrometer ATL resulted in a time-dependent increase in PAR levels which was blocked by NAC. n.s. not significant, ** p
    Goat Anti Rabbit Cy3, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit cy3/product/Jackson Immuno
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit cy3 - by Bioz Stars, 2021-06
    93/100 stars
      Buy from Supplier

    Image Search Results


    Inactivation of the MLL complex prevents the recruitment of MCM2-7 proteins onto chromatin. (A) HCT116 cells were transfected with 50 nM siRNAs of luciferase, Geminin, Geminin+RBBP5, CDT2, CDT2+RBBP5 and RBBP5. The cells were fixed and stained with anti-MCM7 and FITC-conjugated secondary antibodies and counter-stained with DAPI. Scale bars: 50 μm. (B) Proteins from the siRNA-treated cell lysates in A were analyzed by specific antibodies as indicated. (C) Downregulation of WDR5 or RBBP5 reduces the recruitment of MCM proteins to chromatin. Chromatin fractions were isolated from control and WDR5 or RBBP5 siRNA-treated cells and the chromatin-associated MCM proteins were examined by western blotting as indicated. (D) Relative protein levels of MCMs and methylated histone H3K4 on chromatin in C were quantified using Gel-Pro analyzer 4.0. The error bars indicate standard deviation of triplicated samples. The statistical differences of MCM2, MCM3, MCM7, CDT1 and histone H3K4 methylations between control and specific siRNA samples were analyzed using the two-tailed Student's t -test. (E,F) The chromatin immunoprecipitation (ChIP) analysis was performed to locate MCM7 (E) and MCM2 (F) proteins on the DNA replication origin at the Mcm4 gene. Proteins were cross-linked to chromatin and chromatin DNA was sonicated to generate 500-1000 base-pairs (bps) fragments in average length. The ChIP-grade anti-MCM7 and MCM2 antibodies were used for chromatin immunoprecipitation. Cross-linked DNA was released, purified, and analyzed for the enrichment of DNA fragments associated with MCM2 and MCM7 from −4.0 kb to 4.0 kb (kilobase pairs) along the Mcm4 region using various Mcm4 primers and quantitative real time PCR as described in the Materials and Methods. Error bars indicate the standard deviation of triplicated samples. The statistical significance of antibody-enriched specific Mcm4 DNA sequences over the background control IgG binding (fold enrichment) was assessed using the two-tailed Student's t -test.

    Journal: Biology Open

    Article Title: Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases

    doi: 10.1242/bio.019729

    Figure Lengend Snippet: Inactivation of the MLL complex prevents the recruitment of MCM2-7 proteins onto chromatin. (A) HCT116 cells were transfected with 50 nM siRNAs of luciferase, Geminin, Geminin+RBBP5, CDT2, CDT2+RBBP5 and RBBP5. The cells were fixed and stained with anti-MCM7 and FITC-conjugated secondary antibodies and counter-stained with DAPI. Scale bars: 50 μm. (B) Proteins from the siRNA-treated cell lysates in A were analyzed by specific antibodies as indicated. (C) Downregulation of WDR5 or RBBP5 reduces the recruitment of MCM proteins to chromatin. Chromatin fractions were isolated from control and WDR5 or RBBP5 siRNA-treated cells and the chromatin-associated MCM proteins were examined by western blotting as indicated. (D) Relative protein levels of MCMs and methylated histone H3K4 on chromatin in C were quantified using Gel-Pro analyzer 4.0. The error bars indicate standard deviation of triplicated samples. The statistical differences of MCM2, MCM3, MCM7, CDT1 and histone H3K4 methylations between control and specific siRNA samples were analyzed using the two-tailed Student's t -test. (E,F) The chromatin immunoprecipitation (ChIP) analysis was performed to locate MCM7 (E) and MCM2 (F) proteins on the DNA replication origin at the Mcm4 gene. Proteins were cross-linked to chromatin and chromatin DNA was sonicated to generate 500-1000 base-pairs (bps) fragments in average length. The ChIP-grade anti-MCM7 and MCM2 antibodies were used for chromatin immunoprecipitation. Cross-linked DNA was released, purified, and analyzed for the enrichment of DNA fragments associated with MCM2 and MCM7 from −4.0 kb to 4.0 kb (kilobase pairs) along the Mcm4 region using various Mcm4 primers and quantitative real time PCR as described in the Materials and Methods. Error bars indicate the standard deviation of triplicated samples. The statistical significance of antibody-enriched specific Mcm4 DNA sequences over the background control IgG binding (fold enrichment) was assessed using the two-tailed Student's t -test.

    Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated rabbit secondary antibodies (111-097-003) were from Jackson ImmunoResearch Laboratories (West Grove, PA).

    Techniques: Transfection, Luciferase, Staining, Isolation, Western Blot, Methylation, Standard Deviation, Two Tailed Test, Chromatin Immunoprecipitation, Sonication, Purification, Real-time Polymerase Chain Reaction, Binding Assay

    Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P

    Journal: Journal of immunotoxicology

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies

    doi: 10.3109/1547691X.2011.562257

    Figure Lengend Snippet: Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P

    Article Snippet: To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA).

    Techniques: Purification, Mouse Assay, Fluorescence

    Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P

    Journal: Journal of immunotoxicology

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies

    doi: 10.3109/1547691X.2011.562257

    Figure Lengend Snippet: Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P

    Article Snippet: To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA).

    Techniques: Binding Assay, Mouse Assay, Staining, Incubation, In-Cell ELISA

    (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.

    Journal: The Journal of Experimental Medicine

    Article Title: Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

    doi: 10.1084/jem.20050433

    Figure Lengend Snippet: (A) Intracellular staining of GR. PD1.6 cells were treated with 100 nM Dex for 2 h and the mitochondria were stained with 50 nM mitotracker. Rehydrated methanol-fixed cells were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG, and visualized under confocal microscope. Five different cells are presented. (B) Dex induces mitochondrial translocation of GR in PD1.6, but not in PD1.6TEC − cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 2 h before subcellular fractionation using the Oncogene fractionation kit. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and D). (C and D) Data were obtained by two different exposure times. The blots were reprobed with antibodies to α-tubulin (B) and CytoC (D). CytoC could be used as a mitochondrial marker because the cells were harvested before any CytoC release. (C) Sensitivity of PD1.6 and PD1.6TEC − cells to Dex-induced apoptosis. Cells were either untreated or treated with 100 nM Dex for 20 h, and percentage of apoptotic cells was determined as in Fig. 1 A . (D) The mitochondrial fraction is not contaminated by cytosolic or nuclear proteins. Subcellular fractions were run side by side on the same gel and analyzed by Western blotting using antibodies to α-tubulin (A), histone 2B (B), and VDAC (C), which react with cytosolic, nuclear, and mitochondrial fractions, respectively. (E) Dex induces a rapid, temperature-dependent translocation of GR to the nucleus and mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 5 and 15 min at 37°C (lanes 1–3) or 4°C (lanes 4–6) before subcellular fractionation as described in Materials and methods. GR was detected by Western blotting using the PA1-511A antibody to GR (A, C, and E). The blots were reprobed with antibodies to α-tubulin (B), histone H2B (D), and VDAC (F). (F) Dex induces sustained GR translocation to the nucleus and the mitochondria in PD1.6 cells. PD1.6 cells were incubated in the absence or presence of 100 nM Dex for 30 min to 5 h and processed as in E. (G) The mitochondrial GR is also immunoreactive to the M20 and P20 antibodies to GR. The mitochondrial samples 1 and 2 of F were rerun on gel and probed with either M20 or P20 antibodies.

    Article Snippet: Caspase 3 activation was analyzed by incubating rehydrated methanol-fixed cells with antibody to cleaved caspase 3 (Asp 175; Cell Signaling Technologies) followed by FITC-conjugated AffiniPure F(ab)2 -fragment of goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories).

    Techniques: Staining, Incubation, Microscopy, Translocation Assay, Fractionation, Western Blot, Marker

    (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.

    Journal: The Journal of Experimental Medicine

    Article Title: Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis

    doi: 10.1084/jem.20050433

    Figure Lengend Snippet: (A) Sensitivity of various lymphoma and leukemia cell lines to Dex-induced apoptosis. Cells were incubated with 100 nM Dex for 20 h, and the DNA content measured by flow cytometry using propidium iodide. Percentage of subdiploid cells is given. (B) Dose–response to Dex. Cells were incubated with various concentrations of Dex and processed as in A. (C) Caspase 3 activation. Untreated or Dex-treated cells were stained for activated caspase 3 as described in Materials and methods. Percentage of positive cells is given. (D) Expression of mGR on lymphoma and leukemia cells. Untreated cells or cells treated with 100 nM Dex for 2 h were incubated with M20 antibodies to GR and FITC-conjugated goat anti–rabbit IgG (dashed line) or FITC-conjugated goat anti–rabbit IgG only (solid line). The fluorescence intensity was measured by flow cytometry.

    Article Snippet: Caspase 3 activation was analyzed by incubating rehydrated methanol-fixed cells with antibody to cleaved caspase 3 (Asp 175; Cell Signaling Technologies) followed by FITC-conjugated AffiniPure F(ab)2 -fragment of goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories).

    Techniques: Incubation, Flow Cytometry, Cytometry, Activation Assay, Staining, Expressing, Fluorescence

    A nontoxic dose of ATL induces oxidative DNA damage and activates PARP in cancer cells. The PC-3 prostate cancer and NCM460 normal colon epithelial cells, with or without 1 h of preincubation in 10 mM NAC, were treated by 10 μM ATL or vehicle control for the indicated times. a , b Measurement of ROS in PC-3 cells by flow cytometry. Data from three independent experiments were presented as mean ± SD. c , d Measurement of ROS in NCM460 cells by flow cytometry. e , f Immunofluorescent staining of cellular 8-oxoG by Cy3-conjugated avidin. PC-3 cells were treated by 10 μM ATL for 12 h (scale bar: 10 μm). Nuclear 8-oxoG intensity was measured using the ImageJ software and the data were processed by the Prism software. g Representative images of alkaline comet assay. PC-3 cells were treated by vehicle control or 10 μM ATL for 12 h. h The tail moment was defined as percentage of tail DNA × tail length, quantified using the TriTek CometScore software. i Immunofluorescence staining for PAR foci in PC-3 cells treated by 10 μM ATL for 12 h. DNA was counterstained with DAPI (scale bar: 5 μm). j Western blot analysis of PAR in PC-3 cells. Ten micrometer ATL resulted in a time-dependent increase in PAR levels which was blocked by NAC. n.s. not significant, ** p

    Journal: Oncogene

    Article Title: Synergistic lethality between PARP-trapping and alantolactone-induced oxidative DNA damage in homologous recombination-proficient cancer cells

    doi: 10.1038/s41388-020-1191-x

    Figure Lengend Snippet: A nontoxic dose of ATL induces oxidative DNA damage and activates PARP in cancer cells. The PC-3 prostate cancer and NCM460 normal colon epithelial cells, with or without 1 h of preincubation in 10 mM NAC, were treated by 10 μM ATL or vehicle control for the indicated times. a , b Measurement of ROS in PC-3 cells by flow cytometry. Data from three independent experiments were presented as mean ± SD. c , d Measurement of ROS in NCM460 cells by flow cytometry. e , f Immunofluorescent staining of cellular 8-oxoG by Cy3-conjugated avidin. PC-3 cells were treated by 10 μM ATL for 12 h (scale bar: 10 μm). Nuclear 8-oxoG intensity was measured using the ImageJ software and the data were processed by the Prism software. g Representative images of alkaline comet assay. PC-3 cells were treated by vehicle control or 10 μM ATL for 12 h. h The tail moment was defined as percentage of tail DNA × tail length, quantified using the TriTek CometScore software. i Immunofluorescence staining for PAR foci in PC-3 cells treated by 10 μM ATL for 12 h. DNA was counterstained with DAPI (scale bar: 5 μm). j Western blot analysis of PAR in PC-3 cells. Ten micrometer ATL resulted in a time-dependent increase in PAR levels which was blocked by NAC. n.s. not significant, ** p

    Article Snippet: Secondary antibodies include goat anti-mouse-Alexa 488, goat anti-rabbit-Cy3, goat anti-rat-Alexa-488, goat anti-mouse-horseradish peroxidase and goat anti-rabbit-horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA).

    Techniques: Flow Cytometry, Staining, Avidin-Biotin Assay, Software, Alkaline Single Cell Gel Electrophoresis, Immunofluorescence, Western Blot