Proteins were extracted using RIPA lysis buffer (Beyotime) and quantified by bicinchoninic acid (BCA) method following the standard protocol. An equal amount of extracts was treated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and shifted onto a nitrocellulose membrane (Millipore). Subsequently, the membranes were incubated with specific primary antibodies PAX6 (1:1000, ab5790, Abcam) and GADPH (1:1000, ab8245, Abcam), and followed by interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Finally, protein signals were examined using an ECL method.
Ab5790
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab5790 is a high-quality antibody produced by Abcam. It is designed for use in laboratory research applications. The core function of this product is to detect and bind to a specific target molecule. Further details regarding its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab52642, by Abcam (3 mentions)
Ab21624, by Merck Group (3 mentions)
Fluorescence microscope, by Leica (3 mentions)
Ab7751, by Abcam (3 mentions)
Ab34712, by Abcam (2 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Mab4401, by Merck Group (2 mentions)
Ab191181, by Abcam (2 mentions)
Ab22035, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Ab6718, by Abcam (2 mentions)
R10367, by Abcam (2 mentions)
F3165 2 mg, by Merck Group (2 mentions)
Bz x810 microscope, by Keyence (2 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
Af2085, by R&D Systems (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab9482, by Abcam (1 mentions)
17 protocols using ab5790
1
Protein Extraction and Western Blot Analysis
2
Immunofluorescence Staining and Flow Cytometry
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The cells were detached with Versene solution (PanEco, Moscow, Russia) and pelleted by centrifugation at 1800 rpm for 5 min, then washed with HBSS (PanEco, Moscow, Russia), fixed in 4% paraformaldehyde for 10 min, washed with PBS, permeabilized with 70% methanol on ice for 10 min, washed twice with PBS and collected by centrifugation at 1800 pm for 5 min. Fixed cells were incubated with primary antibodies to PAX6 (ab5790, Abcam, Cambridge, UK), glial marker S100b (ab52642, Abcam, Cambridge, UK) or neuronal marker βIII tubulin (ab182070, Abcam, Cambridge, UK) at +4 °C for 12 h; washed with PBS; collected by centrifugation at 1800 rpm for 5 min; and incubated with secondary antibodies (Alexa Fluor 488 conjugated, Invitrogen, Carlsbad, CA, USA) for 60 min in the dark. Fixed cells exposed to secondary antibodies were only used as a negative control for the flow cytometry-based quantification. Stained cells were analyzed on a CyFlow ML flow cytometer using the FloMax software (Partec, Goerlitz, Germany). For evaluation of the number of immunopositive cells, the experiment was repeated at least 3 times.
3
Antibody Characterization for Neurodevelopment
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Primary antibodies for β-actin (#12620), phospho (Thr202/Tyr204) ERK (#4370), and ERK (#9102), were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for glutamic acid decarboxylase 65 (GAD65; SC-377154) and Tbr1 (SC-376258; Western blot) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Neu-N (MAB377) and Tbr2 (AB15894) were from Millipore (Burlington, MA, USA). The antibody for Ki67 (550609) was obtained from BD Pharmingen (San José, CA, USA). Antibodies for Sox2 (ab97959), Tbr1 (ab31941; immunofluorescence), vGlut1 (ab77822) and Pax6 (ab5790) were from Abcam Inc. (Cambridge, MA, USA). Secondary fluorescent antibodies were obtained from Jackson ImmunoResearch Co. Laboratories (West Grove, PA, USA). Polyvinylidene difluoride (PVDF), membranes and molecular weight standards for Western blot were obtained from BIO-RAD (Hercules, CA, USA). The enhanced chemiluminescence (ECL) Western blotting system was from Thermo Fisher Scientific Inc. (Piscataway, NJ, USA). Zinquin, the antibody for γ amino butyric acid (GABA; A2052), and all other reagents were of the highest quality available and were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
4
Immunofluorescence Characterization of Chondrocytes
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Human NP cells grown on cover glass underwent fixation with 4% formalin (20 min) at ambient, permeabilization with 0.1% Triton X-100 and 0.2% Tween-20 in PBS (40 min at ambient), blocking with 2% goat serum (Invitrogen; 1 h), and incubation with anti-collagen-II (1:200; Abcam, Ab34712), anti-Aggrecan (1:500; Abcam, Ab5790), anti-MMP13 (1:50; Abcam, Ab21624), and anti-ADAMT-5 (1:1000; Millipore, MAB4401) primary antibodies, respectively. After washing, the samples further underwent incubation with fluorescein-conjugated secondary antibodies. Images were captured under a fluorescence microscope (Leica).
5
Immunofluorescence Staining of Cerebral Organoids
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Cerebral organoids were fixed with 4% paraformaldehyde in PBS. The samples were embedded in Optimal Cutting Temperature Compound (45,833, Sakura Finetek Japan) and sectioned at a thickness of 10 μm using Leica CM3050 S Cryostat. Organoid sections were permeabilized and blocked with blocking solution (0.05% Triton X-100 and 5% goat serum in PBS). Primary antibodies in blocking solution were then added and incubated at 4 °C for overnight, followed by washing and incubation with secondary antibodies. The following antibodies were used—primary antibodies: rabbit anti-pax6 antibody (ab5790, Abcam), mouse anti-β-tubulin III antibody (ab7751, Abcam), and rat anti-ctip2 antibody (ab18465, Abcam) and secondary antibodies: donkey antirat (ab150155, Abcam), donkey antimouse, (A10036, Invitrogen), and donkey antirabbit, (A21206, Invitrogen). Images were viewed using a confocal microscope (TCS SP8, Leica). The image intensity was adjusted using the ImageJ software (NIH).
6
Immunostaining of Lentivirus-Transduced Brain Sections
7
Pluripotent Stem Cell Characterization
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Human ESCs and iPSCs were cultured on cover glass and fixed by incubating with 4% paraformaldehyde for 20 min at room temperature (RT). Fixed cells were then washed with PBS and permeabilized using a non-ionic detergent (0.1% Triton X-100 and 0.2% Tween-20) in PBS for 40 min at RT. Permeabilized cells were blocked by incubating with 2% goat serum (Invitrogen) for 1 h, washed with PBS containing 0.01% Tween-20 (PBST), and incubated with primary antibody. Primary antibodies used included anti-SOX17 (1:200; R&D Systems; AF1924), anti-PAX6 (1:500; Abcam; Ab5790), anti-NANOG (1:50; Abcam; Ab21624), anti-OCT4 (1:1000; Millipore; MAB4401), and anti-Brachyury (1:50; R&D Systems; AF2085). Cells were then washed with PBST and incubated with the appropriate fluorescein-conjugated secondary antibody. Stained samples were mounted using Vectashield H-1200 mounting media (Vector Laboratories), and images were captured using a fluorescence microscope (Leica). Positive signals in IF images were processed and counted using Image J with a consistent intensity threshold.
8
Immunostaining of Lentivirus-Transduced Brain Sections
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Lentivirus injected brain sections were post-fixed with 4% PFA for 10 min. Then both lentivirus injected and hNPC injected brain sections were washed with PBS three times and incubated 30 min with blocking solution (PBS/0,1% Triton X-100 containing 10% normal goat serum [Sigma-Aldrich]) and then incubated overnight at 4 °C in blocking solution with primary antibodies: mouse anti-Flag (Sigma, F3165–2 MG, 1:1000); rabbit anti-red fluorescence protein (RFP) (Invitrogen, R10367, 1:1000); rabbit anti-PAX6 (Abcam, ab5790, 1:50); mouse anti-Nestin (Abcam, ab22035, 1:100), rabbit anti-Ki67 (Abcam, ab15580, 1:1000) and mouse anti-human nuclear antigen (Abcam, ab191181, 1:1000). Sections were washed with PBS and incubated for 1 h at room temperature with the secondary antibodies: goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher, A31560, 1:500); goat anti-rabbit IgG TRITC (Abcam, ab6718, 1:1000) and goat anti-mouse IgG Alexa Fluor 647 (Invitrogen, A32728, 1:1000) diluted in blocking solution. The sections were washed with PBS and mounted with Vectashield Antifade Mounting Medium (Vector Labs, H-1000). Immunofluorescence was visualized and imaged with a Keyence BZ-X810 microscope.
9
Pluripotent Stem Cell Characterization
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The cells were digested by 0.25% trypsin, collected, and resuspended with 1 mL of 4% paraformaldehyde for 3 min. The cells were resuspended in PBS containing 0.1% Triton X-100 and permeabilized for 30 min. The collected cells were then sealed with 5% sheep serum at 37°C for 30 min, and the supernatant was discarded after centrifugation. The cells were resuspended with primary antibodies against sex determining region Y-box 1 (SOX1; ab109290, Abcam), paired box gene 6 (Pax6; ab5790, Abcam), and octamer-binding protein (Oct4; ab181557, Abcam) at a 1 : 500 dilution with 3% goat serum at 37°C for 1 h. The cells were then treated with secondary antibodies diluted to 1 : 1000 using 3% goat serum for 30 min, after which the fluorescence of the PBS-resuspended cells was measured by flow cytometry.
10
Immunofluorescence Staining of Neural Markers
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Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, United States) for 18 min and washed with PBS solution. Then all the cells were treated with 0.2% Triton X-100 (Sigma-Aldrich, United States) for 8 min. Cells were then blocked with 3% bovine serum albumin (BSA) (Sigma-Aldrich, United States) in PBS solution for 1 h. Then incubated cells with primary antibodies, anti-Pax6 antibody (Abcam, ab5790, United States) or anti-TUJ1 antibody (Abcam, ab78078, United States), were diluted in 1% (w/v) BSA in PBS solution overnight at 4°C. After the incubation, cells were washed with PBS three times and stained with secondary antibodies for 2 h at room temperature.
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