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    Name:
    PBS Phosphate Buffered Saline Tablets
    Description:
    Phosphate Buffered Saline PBS is a balanced salt solution used for a variety of applications including reagent preparation diluting cells for flow cytometry and as a cell culture reagent These PBS tablets are not intended for cell culture as they are not tested for sterility We offer a variety of Gibco PBS formulations for cell culture applications Find the right cell culture product using the media selector tool
    Catalog Number:
    003002
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Cell Analysis|Cellular Imaging|IHC Counterstaining & Mounting|IHC Epitope Retrieval, Blocking & Quenching|IHC Sample Preparation|IHC Staining & Detection|Immunocytochemistry (ICC)|Immunofluorescence (IF)|Immunohistochemistry (IHC)
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    Structured Review

    Thermo Fisher 1x pbs
    Mathematical representation and quantification of contributions of channel depth and air cavity to compliance. ( A ) Mathematical representation of conventional viscometer and microfluidic system. ( a ) Mathematical representation of conventional viscometer. ( b ) Mathematical representation of microfluidic system. ( B ) Evaluation of channel depth ( H ) on compliance. Here, <t>1x</t> PBS as test fluid was infused into a microfluidic system. ( a ) Variations of λ B with respect to H . ( b ) Variations of R WB and C B with respect to H . ( C ) Evaluation of air cavity on compliance.
    Phosphate Buffered Saline PBS is a balanced salt solution used for a variety of applications including reagent preparation diluting cells for flow cytometry and as a cell culture reagent These PBS tablets are not intended for cell culture as they are not tested for sterility We offer a variety of Gibco PBS formulations for cell culture applications Find the right cell culture product using the media selector tool
    https://www.bioz.com/result/1x pbs/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    1x pbs - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Blood Viscoelasticity Measurement Using Interface Variations in Coflowing Streams under Pulsatile Blood Flows"

    Article Title: Blood Viscoelasticity Measurement Using Interface Variations in Coflowing Streams under Pulsatile Blood Flows

    Journal: Micromachines

    doi: 10.3390/mi11030245

    Mathematical representation and quantification of contributions of channel depth and air cavity to compliance. ( A ) Mathematical representation of conventional viscometer and microfluidic system. ( a ) Mathematical representation of conventional viscometer. ( b ) Mathematical representation of microfluidic system. ( B ) Evaluation of channel depth ( H ) on compliance. Here, 1x PBS as test fluid was infused into a microfluidic system. ( a ) Variations of λ B with respect to H . ( b ) Variations of R WB and C B with respect to H . ( C ) Evaluation of air cavity on compliance.
    Figure Legend Snippet: Mathematical representation and quantification of contributions of channel depth and air cavity to compliance. ( A ) Mathematical representation of conventional viscometer and microfluidic system. ( a ) Mathematical representation of conventional viscometer. ( b ) Mathematical representation of microfluidic system. ( B ) Evaluation of channel depth ( H ) on compliance. Here, 1x PBS as test fluid was infused into a microfluidic system. ( a ) Variations of λ B with respect to H . ( b ) Variations of R WB and C B with respect to H . ( C ) Evaluation of air cavity on compliance.

    Techniques Used: Western Blot

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    Article Snippet: .. Investigation of the 24 h post-injection systemic TNF-α and LPS concentration Serum concentrations of TNF-α and LPS were determined 24 h after the peritumoral injection of the first dose of PBS (control), LPS or LPS-NP at high concentrations (1000 µg/mL) using ELISA (eBioscience) and LAL chromogenic endotoxin quantification assay, respectively. .. Microscopic evaluation of the tumor cross-sections For tracking the LPS penetration (both free and nanoparticle-bound) into the tumor after injection, FITC-labeled LPS was utilized in the preparation of both the solution and nanoparticles, which were injected in three corners around the tumor.

    Injection:

    Article Title: A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides
    Article Snippet: .. Investigation of the 24 h post-injection systemic TNF-α and LPS concentration Serum concentrations of TNF-α and LPS were determined 24 h after the peritumoral injection of the first dose of PBS (control), LPS or LPS-NP at high concentrations (1000 µg/mL) using ELISA (eBioscience) and LAL chromogenic endotoxin quantification assay, respectively. .. Microscopic evaluation of the tumor cross-sections For tracking the LPS penetration (both free and nanoparticle-bound) into the tumor after injection, FITC-labeled LPS was utilized in the preparation of both the solution and nanoparticles, which were injected in three corners around the tumor.

    Enzyme-linked Immunosorbent Assay:

    Article Title: A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides
    Article Snippet: .. Investigation of the 24 h post-injection systemic TNF-α and LPS concentration Serum concentrations of TNF-α and LPS were determined 24 h after the peritumoral injection of the first dose of PBS (control), LPS or LPS-NP at high concentrations (1000 µg/mL) using ELISA (eBioscience) and LAL chromogenic endotoxin quantification assay, respectively. .. Microscopic evaluation of the tumor cross-sections For tracking the LPS penetration (both free and nanoparticle-bound) into the tumor after injection, FITC-labeled LPS was utilized in the preparation of both the solution and nanoparticles, which were injected in three corners around the tumor.

    Incubation:

    Article Title: Potent mouse monoclonal antibodies that block SARS-CoV-2 infection
    Article Snippet: Cells were blocked by 1% non-fat skim milk in PBS-T for 10 min, then incubated with 0.5 µg/mL antibody for 1 h at room temperature. .. After three times wash in PBS-T, cells were incubated in 1:500 diluted Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher) and 1 µg/mL DAPI solution for 30 min at room temperature. .. The cover glasses were mounted with Prolong Glass Antifade Mountant (Thermo Fisher) overnight at room temperature before observing.

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    Modification:

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    Cell Culture:

    Article Title: Renal-targeted delivery of triptolide by entrapment in pegylated TRX-20-modified liposomes
    Article Snippet: .. After enzymatic digestions with collagenase IV (0.1% w/v) at 37°C in PBS solution for 20–45 min, the MC suspensions were obtained and cultured in RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum, 2 μg/mL insulin, 300 μg/L transferrin, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified 5% (v/v) CO2 incubator (Thermo Scientific, Marietta, OH, USA). ..

    other:

    Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems
    Article Snippet: The reaction underwent heat inactivation at 75°C for 5 min, and finally washed for 1 hour with 1X PBS.

    Activity Assay:

    Article Title: A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation
    Article Snippet: .. To assess caspase activity, cells were incubated with FWGP or PBS control as above and stained with a Vybrant FAM Poly Caspases Assay Kit (Molecular Probes) according to the manufacturer’s instructions. .. Briefly, 300 μl of cell suspension (1 x 106 cells/ml) were incubated with VAD-FMK FLICA reagent and Hoechst 33342 for the detection of activated caspases 1, 2, 4, 5, 6, 8 and 9, washed and analyzed by flow cytometry as above.

    Staining:

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    Thermo Fisher 1x pbs
    Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with <t>1x</t> PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).
    1x Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

    Journal: bioRxiv

    Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

    doi: 10.1101/2020.05.13.094268

    Figure Lengend Snippet: Demonstration of untargeted ExSeq with C. elegans (A), a Drosophila embryo (B) and the HeLa human cell lin e (C). The first in situ sequencing round is shown and the different colors (blue, magenta, green, and red) reveal the current base of the amplified cDNA (SOLiD sequencing was used). Scale bars: 20, 10 and 30 microns for panels A, B and C, respectively, in post-expansion (e.g., actual size) units. Methods used to generate this figure: Worm fixation and cuticle reduction was adopted from the published Bouin’s tube fixation protocol ( 127 ). The strain used in the figure was CZ1632: juIs76 [unc-25p::GFP + lin-15(+)] II. The strain was maintained at 20°C under standard conditions ( 128 ). The worms were collected from agar plates with M9 buffer (3g KH 2 PO 4 , 6g Na 2 HPO 4 , 5g NaCl, 1ml 1M MgSO 4 , water to 1 liter, sterilized by autoclaving) into a 15 mL tube. The tube was spun down at 1000g for 2 min, and the supernatant was replaced with 10 mL of fresh M9. The M9 wash step was repeated 2 more times. The worms were then transferred to a 1.5 mL tube and spun down to remove as much supernatant as possible without disturbing the worm pellet. The worms were placed on ice for 5 min. 1 mL of Bouin’s Fixative (0.46% picric acid, 4.4% paraformaldehyde, 2.4% acetic acid, 50% methanol, 1.2% 2-mercaptoethanol; as prepared in the published protocol), prepared fresh and pre-chilled to 4°C, was then added. The pellet was resuspended and mixed well. The sample was then placed on a tube rotator and mixed vigorously for 30 min at 25°C, followed by 4 hours of incubation at 4°C. The sample was then washed 3 times with 1mL Borate Triton β-mercaptoethanol solution (BTB; see recipe below); each time the sample was spun down, the supernatant was removed, the buffer was added and the sample was mixed thoroughly. BTB was prepared fresh using 1 mL 40x Borate Buffer Stock (3.1g boric acid, 1g NaOH, water to 50 mL), 1 mL 20% Triton X-100, 0.8 mL 2-mercaptoethanol, and 37.2 mL water. The sample was then further incubated three times, for 1 hour each, in 1 mL fresh BTB on a tube rotator at 25°C. Finally, the sample was washed six times: twice with 1 mL BT (1 mL 40x Borate Buffer Stock, 1 mL 20% Triton X-100, 38 mL water), twice with 1 mL 1x PBST (1x PBS, 0.5% Triton X-100), and twice with 1x PBS. The worms were permeabilized for 1 hour with 0.25% Triton X-100 in 1X PBS at 25°C. Drosophila larvae w1118 (BL#5905) were kindly provided by the lab of Aravinthan DT Samuel (Harvard University). Drosophila were raised in vials or bottles with standard yeast-containing medium at 22°C with alternating 12-h cycles of dark and light. HeLa (ATCC CCL-2) cells were cultured on CultureWell Chambered 16 wells Coverglass (Invitrogen) in D10 medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin–streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin in PBS for 15 min at 25°C, and washed three times with 1× PBS. Fixed cells were then stored in 70% ethanol at 4°C until use. ExSeq experimental procedures for the worms, Drosophila and HeLa cells were performed according to the following Methods sections: ‘RNA anchoring’, ‘Gelling, digestion and expansion’, ‘Re-embedding’, ‘Passivation’, and ‘Library preparation for in situ sequencing’. For the reverse transcription, instead of SSIV, M-MuLV (10U/µl; Enzymatics, cat. no. P7040L) was used for the worms and HeLa cells, whereas Maxima (10U/µl; Thermo Scientific, cat. no. EP0741) was used for Drosophila. Aminoallyl-dUTP was not included in the reverse transcription mix (and therefore the cDNA were not formalin-fixed), and the following primer sequence was used: /5Phos/ACTTCAGCTGCCCCGGGTGAAGANNNNNNNN. For the worms and HeLa cells, 2U/µl CircLigase II were used for circularization. To account for possible self-circularization of the primers, control samples with no reverse transcription enzyme were also processed for the worms, Drosophila, and HeLa cells, and as expected produced only a weak signal with hybridization probe after the library preparation. In situ sequencing was performed manually using the reagents and the enzymatic reactions outlined in the Methods section ‘Automated in situ sequencing’. Imaging was performed on a Zeiss Laser Scanning Confocal (LSM710) with Nikon 40X CFI Apo, water immersion with long working distance, NA 1.15 objective, and excitation light sources and emission filters as in ( 23 ).

    Article Snippet: The reaction underwent heat inactivation at 75°C for 5 min, and finally washed for 1 hour with 1X PBS.

    Techniques: In Situ, Sequencing, Amplification, Incubation, Cell Culture, Produced, Hybridization, Imaging

    Anchoring of cDNA ensures precise spatial capture of cDNA location. In principle, without cDNA anchoring, the cDNA can move after the RNase digestion, as they are no longer linked to the gel in any way. To quantify this effect, we measured the location of the cDNA before the RNase digestion, and then 12 hours after RNase digestion, to create an extreme scenario in which the cDNA could, in principle, move quite a bit. The experiment was done both with and without cDNA anchoring, and performed in cultured hippocampal neurons. (A) BrdU antibody (see Methods at end of the figure caption, below) against cDNA before the RNAse step (green) and 12 hours after the RNase step (red), registered together (yellow overlap). (B) Same as (A), but the cDNA are anchored to the gel before the RNase step. Focusing just on a single soma, (C) and (D), a normalized cross-correlation method using 21×21×13pixel subvolumes from random locations within the volume shown was used to calculate cDNA drifts between the two imaging times. The locations of the 10,000 21×21×13pixel subvolumes were randomly chosen and required to have a minimum signal intensity to ensure sufficient signal-to-noise ratio for the autocorrelation calculation to calculate a correct offset. As shown in Cii and Dii, the direction and magnitude of the offsets are encoded in an RGB image. For each subvolume, the offset of the peak of the normalized cross-correlation is displayed in red for the X-direction offset (black=zero offset, bright red = offset of 10 pixels or 1.7µm), green for Y-direction offset (black=zero offset, bright green = offset of 10 pixels or 1.7µm) and blue for Z-direction offset (black=zero offset, bright blue = offset of 5 pixels or 2.0µm). (E) Only 52% of the subvolumes for the without-cDNA-anchoring condition are within 0.5µm, whereas 99% of the subvolumes match that criteria when cDNA anchoring was used (F). Scale bar: (A, B) 13µm pre-expansion, (C, D) 3µm pre-expansion. The BrdU staining protocol was performed as follows: 5-bromo-2′-deoxyuridine triphosphate was mixed with G, A, C to a concentration of 5mM. This mix was used instead of dNTPs in the reverse transcription reaction with final concentration of 0.25mM. After the reverse transcription, the RNase treatment, and the 12 hour wash in PBS, 5µg/ml anti-BrdU mouse antibody (Anti-Bromodeoxyuridine from mouse IgG1; Roche) in 1x PBS was incubated for 1 hour at room temperature. After washing with 1x PBS, 10µg/ml of goat anti-mouse cy5 antibody (abcam) in 1x PBS was incubated for 1 hour at room temperature. Finally the sample was washed again with 1x PBS. The cDNA anchoring was done as described in the Methods section ‘Library preparation for in situ sequencing’.

    Journal: bioRxiv

    Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

    doi: 10.1101/2020.05.13.094268

    Figure Lengend Snippet: Anchoring of cDNA ensures precise spatial capture of cDNA location. In principle, without cDNA anchoring, the cDNA can move after the RNase digestion, as they are no longer linked to the gel in any way. To quantify this effect, we measured the location of the cDNA before the RNase digestion, and then 12 hours after RNase digestion, to create an extreme scenario in which the cDNA could, in principle, move quite a bit. The experiment was done both with and without cDNA anchoring, and performed in cultured hippocampal neurons. (A) BrdU antibody (see Methods at end of the figure caption, below) against cDNA before the RNAse step (green) and 12 hours after the RNase step (red), registered together (yellow overlap). (B) Same as (A), but the cDNA are anchored to the gel before the RNase step. Focusing just on a single soma, (C) and (D), a normalized cross-correlation method using 21×21×13pixel subvolumes from random locations within the volume shown was used to calculate cDNA drifts between the two imaging times. The locations of the 10,000 21×21×13pixel subvolumes were randomly chosen and required to have a minimum signal intensity to ensure sufficient signal-to-noise ratio for the autocorrelation calculation to calculate a correct offset. As shown in Cii and Dii, the direction and magnitude of the offsets are encoded in an RGB image. For each subvolume, the offset of the peak of the normalized cross-correlation is displayed in red for the X-direction offset (black=zero offset, bright red = offset of 10 pixels or 1.7µm), green for Y-direction offset (black=zero offset, bright green = offset of 10 pixels or 1.7µm) and blue for Z-direction offset (black=zero offset, bright blue = offset of 5 pixels or 2.0µm). (E) Only 52% of the subvolumes for the without-cDNA-anchoring condition are within 0.5µm, whereas 99% of the subvolumes match that criteria when cDNA anchoring was used (F). Scale bar: (A, B) 13µm pre-expansion, (C, D) 3µm pre-expansion. The BrdU staining protocol was performed as follows: 5-bromo-2′-deoxyuridine triphosphate was mixed with G, A, C to a concentration of 5mM. This mix was used instead of dNTPs in the reverse transcription reaction with final concentration of 0.25mM. After the reverse transcription, the RNase treatment, and the 12 hour wash in PBS, 5µg/ml anti-BrdU mouse antibody (Anti-Bromodeoxyuridine from mouse IgG1; Roche) in 1x PBS was incubated for 1 hour at room temperature. After washing with 1x PBS, 10µg/ml of goat anti-mouse cy5 antibody (abcam) in 1x PBS was incubated for 1 hour at room temperature. Finally the sample was washed again with 1x PBS. The cDNA anchoring was done as described in the Methods section ‘Library preparation for in situ sequencing’.

    Article Snippet: The reaction underwent heat inactivation at 75°C for 5 min, and finally washed for 1 hour with 1X PBS.

    Techniques: Cell Culture, Imaging, BrdU Staining, Concentration Assay, Incubation, In Situ, Sequencing

    Effects of reduction level on free sulfhydryl content of Igs and vaccine efficacy. (a) Susceptibility of human and murine monoclonal Ig preparations to DTT reduction. Human IgG1-R, IgG1-T, and murine IgG2a (A20 Id) were exposed to varying concentrations of DTT (0.01, 0.1, 1, 5, 10, 50, 100, 300 mM) for 1 hour at 37°C, followed by buffer exchange into 1X PBS plus 0.1M EDTA, and free sulfhydryl (SH) content was determined via Ellman's test. (b) Effect of Id reduction conditions before maleimide conjugation on Id-KLH vaccine efficacy in vivo . A20 Id was reduced using 0.1, 10, or 300 mM DTT, then conjugated to maleimide-activated KLH. Groups of mice (n = 8) were inoculated s.c. with 1×10 5 A20 tumor cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or control treatment with HBSS, and followed for survival. Data are representative of two independent experiments.

    Journal: Molecular immunology

    Article Title: Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

    doi: 10.1016/j.molimm.2008.10.020

    Figure Lengend Snippet: Effects of reduction level on free sulfhydryl content of Igs and vaccine efficacy. (a) Susceptibility of human and murine monoclonal Ig preparations to DTT reduction. Human IgG1-R, IgG1-T, and murine IgG2a (A20 Id) were exposed to varying concentrations of DTT (0.01, 0.1, 1, 5, 10, 50, 100, 300 mM) for 1 hour at 37°C, followed by buffer exchange into 1X PBS plus 0.1M EDTA, and free sulfhydryl (SH) content was determined via Ellman's test. (b) Effect of Id reduction conditions before maleimide conjugation on Id-KLH vaccine efficacy in vivo . A20 Id was reduced using 0.1, 10, or 300 mM DTT, then conjugated to maleimide-activated KLH. Groups of mice (n = 8) were inoculated s.c. with 1×10 5 A20 tumor cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or control treatment with HBSS, and followed for survival. Data are representative of two independent experiments.

    Article Snippet: A20 maleimide Id-KLH conjugates (1 mg/ml Id) were stored for 6 months at 4°C or -80°C in 1X PBS, pH 7.2, and the integrity of the maleimide linkage was compared to freshly conjugated maleimide Id-KLH by SDS-PAGE analysis under non-reducing conditions on a 12% Precise Protein Gel (Pierce).

    Techniques: Buffer Exchange, Conjugation Assay, In Vivo, Mouse Assay

    Stability and immunologic potency of maleimide Id-KLH conjugates is maintained after long-term frozen storage. A20 conjugates (1mg/ml Id in 1X PBS, pH 7.2) were stored for 6 months at 4°C or -80°C, and compared with freshly conjugated Id-KLH. (a) Long-term stability of frozen maleimide Id-KLH. Conjugates (10 μg) were subjected to non-reducing SDS-PAGE alongside 5 μg of free A20 Id. The absence of free Id in the freshly-conjugated or -80°C stored lane indicates that Id is completely conjugated to the carrier protein and thus stable under these storage conditions. Some free Id is seen in the sample stored at 4°C for 6 months, indicating that the Id-KLH linkage is less stable under these conditions. (b) In vivo efficacy of maleimide Id-KLH conjugates after long term storage. Mice (n = 8) were inoculated s.c with 1×10 5 A20 cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or HBSS, and followed for survival.

    Journal: Molecular immunology

    Article Title: Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

    doi: 10.1016/j.molimm.2008.10.020

    Figure Lengend Snippet: Stability and immunologic potency of maleimide Id-KLH conjugates is maintained after long-term frozen storage. A20 conjugates (1mg/ml Id in 1X PBS, pH 7.2) were stored for 6 months at 4°C or -80°C, and compared with freshly conjugated Id-KLH. (a) Long-term stability of frozen maleimide Id-KLH. Conjugates (10 μg) were subjected to non-reducing SDS-PAGE alongside 5 μg of free A20 Id. The absence of free Id in the freshly-conjugated or -80°C stored lane indicates that Id is completely conjugated to the carrier protein and thus stable under these storage conditions. Some free Id is seen in the sample stored at 4°C for 6 months, indicating that the Id-KLH linkage is less stable under these conditions. (b) In vivo efficacy of maleimide Id-KLH conjugates after long term storage. Mice (n = 8) were inoculated s.c with 1×10 5 A20 cells on day 0, and on day 4 began 3 weekly immunizations with the respective conjugates plus GM-CSF, or HBSS, and followed for survival.

    Article Snippet: A20 maleimide Id-KLH conjugates (1 mg/ml Id) were stored for 6 months at 4°C or -80°C in 1X PBS, pH 7.2, and the integrity of the maleimide linkage was compared to freshly conjugated maleimide Id-KLH by SDS-PAGE analysis under non-reducing conditions on a 12% Precise Protein Gel (Pierce).

    Techniques: SDS Page, In Vivo, Mouse Assay

    Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a)  Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS.  (b)  Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA  (c,d)  Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p

    Journal: Biomaterials

    Article Title: Scanningless and continuous 3D bioprinting of human tissues with decellularized extracellular matrix

    doi: 10.1016/j.biomaterials.2018.12.009

    Figure Lengend Snippet: Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a) Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS. (b) Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA (c,d) Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p

    Article Snippet: After incubation, samples were rinsed three times in 1X PBS and immersed in 1X PBS supplemented with 0.05% (v/v) sodium azide (Cat. # 14314–09, Alfa Aesar) prior to taking fluorescent images with an Olympus FV1000 microscope.

    Techniques: Construct, Standard Deviation