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Bio-Rad tris buffered saline tbs
Tris Buffered Saline Tbs, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 97 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tris buffered saline tbs - by Bioz Stars, 2021-05
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Article Title: Inhibition of Mitofusin-2 Promotes Cardiac Fibroblast Activation via the PERK/ATF4 Pathway and Reactive Oxygen Species
Article Snippet: After centrifugation at 16000 × g for 10 min (4°C), the protein concentrations were subsequently determined using the BCA Protein Assay Kit (Thermo Fisher scientific). .. Then the protein samples (25 μ g) were subjected to SDS-PAGE, transferred to a PVDF membrane (Millipore, Bedford, MA), blocked with 5% skim milk in Tris-buffered saline and Tween 20 (TBST) solution for 2 h, and probed with corresponding primary antibodies at 4°C overnight, and HRP-conjugated secondary antibodies (1 : 3000) were incubated for 1 h. The antigen-antibody complexes were detected by enhanced chemiluminescence (ECL) (Bio-Rad). .. Images of the Western blot assay were carried out and analyzed using ChemiScope 6000 (CLINX, China).

Incubation:

Article Title: Inhibition of Mitofusin-2 Promotes Cardiac Fibroblast Activation via the PERK/ATF4 Pathway and Reactive Oxygen Species
Article Snippet: After centrifugation at 16000 × g for 10 min (4°C), the protein concentrations were subsequently determined using the BCA Protein Assay Kit (Thermo Fisher scientific). .. Then the protein samples (25 μ g) were subjected to SDS-PAGE, transferred to a PVDF membrane (Millipore, Bedford, MA), blocked with 5% skim milk in Tris-buffered saline and Tween 20 (TBST) solution for 2 h, and probed with corresponding primary antibodies at 4°C overnight, and HRP-conjugated secondary antibodies (1 : 3000) were incubated for 1 h. The antigen-antibody complexes were detected by enhanced chemiluminescence (ECL) (Bio-Rad). .. Images of the Western blot assay were carried out and analyzed using ChemiScope 6000 (CLINX, China).

Article Title: Combinatorial interactions of genetic variants in human cardiomyopathy
Article Snippet: Lysates underwent gel electrophoresis on a 10–20% Tris glycine SDS–PAGE gel (Novex; EC6135), which was then transferred to polyvinylidene difluoride membrane (Immobilon; IPVH0010) for immunoblotting. .. Blots were blocked in 5% milk in Tris-buffered saline with 1% Tween (TBST), incubated with primary antibody in 5% milk in TBST, washed in TBST and imaged using Clarity Western ECL substrate (Bio-Rad, 170–5060). .. The antibodies used include: anti-vinculin (Sigma, V9131), anti-N-terminal vinculin (Santa Cruz Biotechnology, sc-5573), anti-tropomyosin 1 (Abcam, EPR5159), anti-tubulin (Sigma, T5168) and anti-GAPDH (GeneTex, GTX100118 or Santa Cruz Biotechnology; sc-32233).

Article Title: Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7
Article Snippet: After denaturing at 100°C for 10 min, protein samples were separated on a SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF, Bio-Rad) membrane. .. After blocking with 5% skimmed milk in Tris-Buffered Saline containing 0.5% (v/v) Tween-20 (TBST), the membrane was incubated with primary antibody at 4°C overnight. .. The commercial antibodies used as primary antibody were anti-AKAP12 (ab49849, 1:1000; Abcam, Cambridge, MA, United States), anti-DUSP5 (ab200708,1:1000; Abcam, Cambridge, MA, United States) and anti-beta actin (abs137975,1:5000; Absin, Shanghai, China).

Article Title: Arrestin-3 interaction with maternal embryonic leucine-zipper kinase
Article Snippet: The beads were then washed with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate). .. The membranes were then blocked in a 20 mL solution of 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h. Then, the membranes were washed three times 5 min each in TBS-T before overnight incubation with prey protein (0.2 μM MELK1−326,T167E or 0.3 μM arrestin-31−393) in binding buffer (MELK: 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM DTT; arrestin-3: 20 mM MOPS pH 7.5, 150 mM NaCl, 2 mM TCEP). .. Both cell lysates and co-immunoprecipitated samples were subjected to Western blotting using antibodies against arrestin (1:10,000; polyclonal rabbit F431 [ ]) and MELK (1:1,000; F3165 Sigma).

Western Blot:

Article Title: Combinatorial interactions of genetic variants in human cardiomyopathy
Article Snippet: Lysates underwent gel electrophoresis on a 10–20% Tris glycine SDS–PAGE gel (Novex; EC6135), which was then transferred to polyvinylidene difluoride membrane (Immobilon; IPVH0010) for immunoblotting. .. Blots were blocked in 5% milk in Tris-buffered saline with 1% Tween (TBST), incubated with primary antibody in 5% milk in TBST, washed in TBST and imaged using Clarity Western ECL substrate (Bio-Rad, 170–5060). .. The antibodies used include: anti-vinculin (Sigma, V9131), anti-N-terminal vinculin (Santa Cruz Biotechnology, sc-5573), anti-tropomyosin 1 (Abcam, EPR5159), anti-tubulin (Sigma, T5168) and anti-GAPDH (GeneTex, GTX100118 or Santa Cruz Biotechnology; sc-32233).

other:

Article Title: Synthesis and Characterization of Novel Resveratrol Butyrate Esters That Have the Ability to Prevent Fat Accumulation in a Liver Cell Culture Model
Article Snippet: Next, the membrane was blocked with 5% non-fat dry milk in Tris-buffered saline, 0.1% Tween (TBST) and shaken continuously for 2 h at room temperature.

Article Title: ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile
Article Snippet: Blots were blocked using 5% milk in tris-buffered saline and polysorbate 20 (TBS-T) for an hour at room temperature.

Article Title: mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis.
Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 1% Tween-20 for 1 hr at room temperature.

Blocking Assay:

Article Title: Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7
Article Snippet: After denaturing at 100°C for 10 min, protein samples were separated on a SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF, Bio-Rad) membrane. .. After blocking with 5% skimmed milk in Tris-Buffered Saline containing 0.5% (v/v) Tween-20 (TBST), the membrane was incubated with primary antibody at 4°C overnight. .. The commercial antibodies used as primary antibody were anti-AKAP12 (ab49849, 1:1000; Abcam, Cambridge, MA, United States), anti-DUSP5 (ab200708,1:1000; Abcam, Cambridge, MA, United States) and anti-beta actin (abs137975,1:5000; Absin, Shanghai, China).

Binding Assay:

Article Title: Arrestin-3 interaction with maternal embryonic leucine-zipper kinase
Article Snippet: The beads were then washed with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate). .. The membranes were then blocked in a 20 mL solution of 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h. Then, the membranes were washed three times 5 min each in TBS-T before overnight incubation with prey protein (0.2 μM MELK1−326,T167E or 0.3 μM arrestin-31−393) in binding buffer (MELK: 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM DTT; arrestin-3: 20 mM MOPS pH 7.5, 150 mM NaCl, 2 mM TCEP). .. Both cell lysates and co-immunoprecipitated samples were subjected to Western blotting using antibodies against arrestin (1:10,000; polyclonal rabbit F431 [ ]) and MELK (1:1,000; F3165 Sigma).

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  • 99
    Bio-Rad rat anti mouse cd68 antibody
    Expression of mRNA for 5-HT 3 receptors in muscle layer, quantification of 5-HT 3 receptors and <t>CD68</t> double-positive cells in intestine of control mice or POI mice.and double staining for 5-HT 3 receptors and CD68 in the serosal surface of intestinal wall
    Rat Anti Mouse Cd68 Antibody, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti mouse cd68 antibody/product/Bio-Rad
    Average 99 stars, based on 1 article reviews
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    97
    Bio-Rad tris buffered saline
    The general characterization of female Esr1 -Q mice. All tissues were collected from the adult mice (n = 5 or n=10). ( A ) ERα protein expression. Cytoplasmic and nuclear fractions of samples were extracted from frozen tissues using Nuclear and Cytoplasmic Extraction Reagents. Ten micrograms of cytoplasmic or nuclear extracted proteins were loaded into a 4% to 20% gradient acrylamide gel and run for 40 minutes. Gels were transferred onto nitrocellulose membranes. After blocking using 5% <t>milk/TBS-T</t> buffer for an hour, the blots were incubated overnight with primary ERα antibody (1:1000; MC-20) or GAPDH antibody (1:2000; LF-335) in 5% milk/TBS-T buffer at 4°C. The blots were washed incubated with IRdye 800CW goat anti-mouse or anti-rabbit fluorescent secondary antibody (1:10 000) in 5% milk/TBS-T buffer for an hour at room temperature. The signals were imaged using the Li-Cor Odyssey Fc system. ( B ) RNA expression. Total RNA samples were extracted from frozen tissues using Trizol. The mRNA levels of genes were measured using SYBR green assays. The experiments were repeated 3 times, and results are presented as the fold increase calculated relative to the vehicle of WT ERα ± SE. ( C ) Uterine weight. ( D ) Histology. Mouse tissues were placed immediately into 10% neutral-buffered formalin after euthanasia and then processed and paraffin was embedded once the solution remained clear in 70% (w/v) ethanol for several hours. Tissue was sectioned (7 µm) and placed onto charged-glass microscope slides and stained with hematoxylin and eosin for histopathology analysis. ( E ) Body weight. αERKO, ERα knock-out; cyto, cytoplasmic; ERα, estrogen receptor-α; ext, extraction; Nuc, nuclear; RNA, ribonucleic acid; SE, standard error; TBS-T, <t>tris-buffered</t> saline and polysorbate 20; UT, uterine; WT, wild type.
    Tris Buffered Saline, supplied by Bio-Rad, 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/tris buffered saline/product/Bio-Rad
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris buffered saline - by Bioz Stars, 2021-05
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    97
    Bio-Rad rabbit anti cd31
    Representative and quantitative immunohistochemistry of the peri-infarct cortex before the bregma (coordinate + 0.58) in normal WT, normal sEH KO, MCAO WT, MCAO sEH KO, MCAO with vehicle treatment and MCAO with AUDA treatment mice (n = 2 for normal groups, n = 5–6 for all groups subjected to MCAO). ( A – F , S ) The number of <t>CD31-positive</t> endothelial cells significantly increased in the sEH KO before and after MCAO and post-MCAO AUDA-treated mice, while ( G – L ) Note the increased phosphorylated TrkB (p-TrkB) immunoreactivity that partially co-localized with NeuN-positive neurons, particularly in the sEH KO and AUDA-treated mice (arrows) ( T ). The percentage of NeuN- and p-TrkB-positive double labeling in the neuronal population was significantly increased compared to that in the corresponding controls. ( M – R , U – V ) GFAP-positive astrogliosis and Iba1-positive microglial infiltration decreased in post-MCAO sEH KO and AUDA treatment groups relative to that in the corresponding controls. The scale bar represents 50 µm. * p
    Rabbit Anti Cd31, supplied by Bio-Rad, 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/rabbit anti cd31/product/Bio-Rad
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    99
    Bio-Rad immun star westernc kit
    Detection of full-length acGFP restoration by western blot analysis. After co-transfection of target molecule and either RTM 2 (lane 6), 4 (lane 7), or 3 (lane 4), acGFP protein (27 kDa) was detected by western blot analysis. As positive control, total-cell extract from acGFP control vector (acGFP cloned in pcDNA 3.1D/V5-HIS vector, Invitrogen) transfected HEK293AD cells was also included (lane 2). Lane 3 demonstrates a significant increase (∼3-fold) in the amount of acGFP protein in acGFP positive cells collected by FACS sorting of HEK293AD cells co-transfected with RTM 2 and target expression plasmids (lane 3) compared to total cell extract from co-transfected HEK293AD cells without sorting (lane 6). No detectable acGFP expression was visible after single transfection of target molecule (lane 11), RTM 4 (lane 10) or RTM 2 (lane 9) into HEK293AD cells. No expression of acGFP was detected by western blot analysis of HEK293AD cells co-transfected with target molecule and either RTM 1 (lane 5) or RTM 0 (which is missing a specific BD for intron 52, lane 8). Annexin I (37 kDa) was included as the loading control for this experiment. Lane 1: Precision Plus Protein <t>WesternC</t> Standards (Biorad).
    Immun Star Westernc Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of mRNA for 5-HT 3 receptors in muscle layer, quantification of 5-HT 3 receptors and CD68 double-positive cells in intestine of control mice or POI mice.and double staining for 5-HT 3 receptors and CD68 in the serosal surface of intestinal wall

    Journal: British Journal of Pharmacology

    Article Title: Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus

    doi: 10.1111/bph.13006

    Figure Lengend Snippet: Expression of mRNA for 5-HT 3 receptors in muscle layer, quantification of 5-HT 3 receptors and CD68 double-positive cells in intestine of control mice or POI mice.and double staining for 5-HT 3 receptors and CD68 in the serosal surface of intestinal wall

    Article Snippet: Fixed whole-mount preparations were washed twice with TBS for 30 min and then permeabilized with 0.2% Triton-X-100 and 2% BSA in TBS for 2 h. The permeabilized preparations were rinsed with 2% BSA in TBS for 30 min, incubated with 1:500 diluted rat anti-mouse CD68 antibody (Serotec, Ltd., Oxford, UK) in TBS with 2% BSA at 4°C overnight and then washed for 2 h in TBS.

    Techniques: Expressing, Mouse Assay, Double Staining

    Effects of ondansetron (Ond) on macrophage and neutrophil infiltrations into myenteric plexus region induced by IM. Typical images of CD68-positive macrophages and MPO-positive neutrophils in myenteric plexus region at 24 h after IM are shown

    Journal: British Journal of Pharmacology

    Article Title: Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus

    doi: 10.1111/bph.13006

    Figure Lengend Snippet: Effects of ondansetron (Ond) on macrophage and neutrophil infiltrations into myenteric plexus region induced by IM. Typical images of CD68-positive macrophages and MPO-positive neutrophils in myenteric plexus region at 24 h after IM are shown

    Article Snippet: Fixed whole-mount preparations were washed twice with TBS for 30 min and then permeabilized with 0.2% Triton-X-100 and 2% BSA in TBS for 2 h. The permeabilized preparations were rinsed with 2% BSA in TBS for 30 min, incubated with 1:500 diluted rat anti-mouse CD68 antibody (Serotec, Ltd., Oxford, UK) in TBS with 2% BSA at 4°C overnight and then washed for 2 h in TBS.

    Techniques:

    Double-staining for 5-HT 3 R and CD68-positive macrophages in ileum of mice. (A) Examination of specificity of anti-5-HT 3 receptor antibody. Upper panel or lower panel shows immunohistochemistry by anti-5-HT 3 receptor antibody in ileum of wild-type mice

    Journal: British Journal of Pharmacology

    Article Title: Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus

    doi: 10.1111/bph.13006

    Figure Lengend Snippet: Double-staining for 5-HT 3 R and CD68-positive macrophages in ileum of mice. (A) Examination of specificity of anti-5-HT 3 receptor antibody. Upper panel or lower panel shows immunohistochemistry by anti-5-HT 3 receptor antibody in ileum of wild-type mice

    Article Snippet: Fixed whole-mount preparations were washed twice with TBS for 30 min and then permeabilized with 0.2% Triton-X-100 and 2% BSA in TBS for 2 h. The permeabilized preparations were rinsed with 2% BSA in TBS for 30 min, incubated with 1:500 diluted rat anti-mouse CD68 antibody (Serotec, Ltd., Oxford, UK) in TBS with 2% BSA at 4°C overnight and then washed for 2 h in TBS.

    Techniques: Double Staining, Mouse Assay, Immunohistochemistry

    The general characterization of female Esr1 -Q mice. All tissues were collected from the adult mice (n = 5 or n=10). ( A ) ERα protein expression. Cytoplasmic and nuclear fractions of samples were extracted from frozen tissues using Nuclear and Cytoplasmic Extraction Reagents. Ten micrograms of cytoplasmic or nuclear extracted proteins were loaded into a 4% to 20% gradient acrylamide gel and run for 40 minutes. Gels were transferred onto nitrocellulose membranes. After blocking using 5% milk/TBS-T buffer for an hour, the blots were incubated overnight with primary ERα antibody (1:1000; MC-20) or GAPDH antibody (1:2000; LF-335) in 5% milk/TBS-T buffer at 4°C. The blots were washed incubated with IRdye 800CW goat anti-mouse or anti-rabbit fluorescent secondary antibody (1:10 000) in 5% milk/TBS-T buffer for an hour at room temperature. The signals were imaged using the Li-Cor Odyssey Fc system. ( B ) RNA expression. Total RNA samples were extracted from frozen tissues using Trizol. The mRNA levels of genes were measured using SYBR green assays. The experiments were repeated 3 times, and results are presented as the fold increase calculated relative to the vehicle of WT ERα ± SE. ( C ) Uterine weight. ( D ) Histology. Mouse tissues were placed immediately into 10% neutral-buffered formalin after euthanasia and then processed and paraffin was embedded once the solution remained clear in 70% (w/v) ethanol for several hours. Tissue was sectioned (7 µm) and placed onto charged-glass microscope slides and stained with hematoxylin and eosin for histopathology analysis. ( E ) Body weight. αERKO, ERα knock-out; cyto, cytoplasmic; ERα, estrogen receptor-α; ext, extraction; Nuc, nuclear; RNA, ribonucleic acid; SE, standard error; TBS-T, tris-buffered saline and polysorbate 20; UT, uterine; WT, wild type.

    Journal: Endocrinology

    Article Title: ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile

    doi: 10.1210/endocr/bqaa050

    Figure Lengend Snippet: The general characterization of female Esr1 -Q mice. All tissues were collected from the adult mice (n = 5 or n=10). ( A ) ERα protein expression. Cytoplasmic and nuclear fractions of samples were extracted from frozen tissues using Nuclear and Cytoplasmic Extraction Reagents. Ten micrograms of cytoplasmic or nuclear extracted proteins were loaded into a 4% to 20% gradient acrylamide gel and run for 40 minutes. Gels were transferred onto nitrocellulose membranes. After blocking using 5% milk/TBS-T buffer for an hour, the blots were incubated overnight with primary ERα antibody (1:1000; MC-20) or GAPDH antibody (1:2000; LF-335) in 5% milk/TBS-T buffer at 4°C. The blots were washed incubated with IRdye 800CW goat anti-mouse or anti-rabbit fluorescent secondary antibody (1:10 000) in 5% milk/TBS-T buffer for an hour at room temperature. The signals were imaged using the Li-Cor Odyssey Fc system. ( B ) RNA expression. Total RNA samples were extracted from frozen tissues using Trizol. The mRNA levels of genes were measured using SYBR green assays. The experiments were repeated 3 times, and results are presented as the fold increase calculated relative to the vehicle of WT ERα ± SE. ( C ) Uterine weight. ( D ) Histology. Mouse tissues were placed immediately into 10% neutral-buffered formalin after euthanasia and then processed and paraffin was embedded once the solution remained clear in 70% (w/v) ethanol for several hours. Tissue was sectioned (7 µm) and placed onto charged-glass microscope slides and stained with hematoxylin and eosin for histopathology analysis. ( E ) Body weight. αERKO, ERα knock-out; cyto, cytoplasmic; ERα, estrogen receptor-α; ext, extraction; Nuc, nuclear; RNA, ribonucleic acid; SE, standard error; TBS-T, tris-buffered saline and polysorbate 20; UT, uterine; WT, wild type.

    Article Snippet: Blots were blocked using 5% milk in tris-buffered saline and polysorbate 20 (TBS-T) for an hour at room temperature.

    Techniques: Mouse Assay, Expressing, Acrylamide Gel Assay, Blocking Assay, Incubation, RNA Expression, SYBR Green Assay, Microscopy, Staining, Histopathology, Knock-Out

    Representative and quantitative immunohistochemistry of the peri-infarct cortex before the bregma (coordinate + 0.58) in normal WT, normal sEH KO, MCAO WT, MCAO sEH KO, MCAO with vehicle treatment and MCAO with AUDA treatment mice (n = 2 for normal groups, n = 5–6 for all groups subjected to MCAO). ( A – F , S ) The number of CD31-positive endothelial cells significantly increased in the sEH KO before and after MCAO and post-MCAO AUDA-treated mice, while ( G – L ) Note the increased phosphorylated TrkB (p-TrkB) immunoreactivity that partially co-localized with NeuN-positive neurons, particularly in the sEH KO and AUDA-treated mice (arrows) ( T ). The percentage of NeuN- and p-TrkB-positive double labeling in the neuronal population was significantly increased compared to that in the corresponding controls. ( M – R , U – V ) GFAP-positive astrogliosis and Iba1-positive microglial infiltration decreased in post-MCAO sEH KO and AUDA treatment groups relative to that in the corresponding controls. The scale bar represents 50 µm. * p

    Journal: Scientific Reports

    Article Title: Blockade of soluble epoxide hydrolase attenuates post-ischemic neuronal hyperexcitation and confers resilience against stroke with TrkB activation

    doi: 10.1038/s41598-017-18558-6

    Figure Lengend Snippet: Representative and quantitative immunohistochemistry of the peri-infarct cortex before the bregma (coordinate + 0.58) in normal WT, normal sEH KO, MCAO WT, MCAO sEH KO, MCAO with vehicle treatment and MCAO with AUDA treatment mice (n = 2 for normal groups, n = 5–6 for all groups subjected to MCAO). ( A – F , S ) The number of CD31-positive endothelial cells significantly increased in the sEH KO before and after MCAO and post-MCAO AUDA-treated mice, while ( G – L ) Note the increased phosphorylated TrkB (p-TrkB) immunoreactivity that partially co-localized with NeuN-positive neurons, particularly in the sEH KO and AUDA-treated mice (arrows) ( T ). The percentage of NeuN- and p-TrkB-positive double labeling in the neuronal population was significantly increased compared to that in the corresponding controls. ( M – R , U – V ) GFAP-positive astrogliosis and Iba1-positive microglial infiltration decreased in post-MCAO sEH KO and AUDA treatment groups relative to that in the corresponding controls. The scale bar represents 50 µm. * p

    Article Snippet: The slides were incubated in a blocking solution containing 3% donkey serum albumin (Abcam) and 0.3% Triton X-100 (Sigma-Aldrich) in TBS for 1 hour at room temperature and immunostained with primary antibodies including rabbit anti-CD31 (1:200, BioRad, MCA23886A), anti-p-TrkB (1:200, Abcam, ab109684), anti-NeuN (1:200, Abcam, ab104224), anti-GFAP (1:500, Abcam, ab7260), and anti-Iba1 (1:200, Novus, NB100-1028) overnight at 4 °C.

    Techniques: Immunohistochemistry, Mouse Assay, Labeling

    Detection of full-length acGFP restoration by western blot analysis. After co-transfection of target molecule and either RTM 2 (lane 6), 4 (lane 7), or 3 (lane 4), acGFP protein (27 kDa) was detected by western blot analysis. As positive control, total-cell extract from acGFP control vector (acGFP cloned in pcDNA 3.1D/V5-HIS vector, Invitrogen) transfected HEK293AD cells was also included (lane 2). Lane 3 demonstrates a significant increase (∼3-fold) in the amount of acGFP protein in acGFP positive cells collected by FACS sorting of HEK293AD cells co-transfected with RTM 2 and target expression plasmids (lane 3) compared to total cell extract from co-transfected HEK293AD cells without sorting (lane 6). No detectable acGFP expression was visible after single transfection of target molecule (lane 11), RTM 4 (lane 10) or RTM 2 (lane 9) into HEK293AD cells. No expression of acGFP was detected by western blot analysis of HEK293AD cells co-transfected with target molecule and either RTM 1 (lane 5) or RTM 0 (which is missing a specific BD for intron 52, lane 8). Annexin I (37 kDa) was included as the loading control for this experiment. Lane 1: Precision Plus Protein WesternC Standards (Biorad).

    Journal: Nucleic Acids Research

    Article Title: A novel screening system improves genetic correction by internal exon replacement

    doi: 10.1093/nar/gkr465

    Figure Lengend Snippet: Detection of full-length acGFP restoration by western blot analysis. After co-transfection of target molecule and either RTM 2 (lane 6), 4 (lane 7), or 3 (lane 4), acGFP protein (27 kDa) was detected by western blot analysis. As positive control, total-cell extract from acGFP control vector (acGFP cloned in pcDNA 3.1D/V5-HIS vector, Invitrogen) transfected HEK293AD cells was also included (lane 2). Lane 3 demonstrates a significant increase (∼3-fold) in the amount of acGFP protein in acGFP positive cells collected by FACS sorting of HEK293AD cells co-transfected with RTM 2 and target expression plasmids (lane 3) compared to total cell extract from co-transfected HEK293AD cells without sorting (lane 6). No detectable acGFP expression was visible after single transfection of target molecule (lane 11), RTM 4 (lane 10) or RTM 2 (lane 9) into HEK293AD cells. No expression of acGFP was detected by western blot analysis of HEK293AD cells co-transfected with target molecule and either RTM 1 (lane 5) or RTM 0 (which is missing a specific BD for intron 52, lane 8). Annexin I (37 kDa) was included as the loading control for this experiment. Lane 1: Precision Plus Protein WesternC Standards (Biorad).

    Article Snippet: After washing with TBS-T the blot was visualized using the Immun-Star WesternC Kit (Biorad).

    Techniques: Western Blot, Cotransfection, Positive Control, Plasmid Preparation, Transfection, FACS, Expressing