Kidneys were lysed into RIPA-buffer for 10 minutes on ice. Protein concentration was determined with bichloroacetic acid protein assay kit (Pierce, Rockford, IL). Equal amounts (30–50 μg) of protein extracts were loaded and separated by SDS-PAGE using 7.5%, 10%, 12.5%, and 15% polyacrylamide gels by 100 V for 140 min and then transferred to the polyvinylidene fluoride membrane for 3 hours (Millipore, Bedford, MA). Membranes were blocked with 4% skimmed milk in TBS-0.5% Tween 20 for 1 hour and then incubated with primary antibody anti-β-actin (Sigma, USA), anti-CD68 (Abcam), anti-TGF-β (Abcam, UK), CD206 (Abcam, UK), anti-WT-1 (Abcam, UK), anti-caspase-3 (Cell Signaling, Danvers, MA), anti-collagen-I (Abcam, UK), anti-HO-1 (Abcam), and PCNA (Abcam, UK) for 4°C overnight. The membranes were washed using TBS-0.5% Tween 20 and incubated with secondary antibodies, peroxidase-conjugated anti-rabbit (1 : 40000, Sigma, USA) and anti-mouse (1 : 40000, Sigma, USA) for 1 hour at room temperature. The signals on the membrane were detected using Immobilon Western Chemiluminescent HRP Substrate Kit (GE, USA) and exposed to X-ray film (GE, USA) for autoradiogram.
Anti wt1
Manufactured by Abcam
Sourced in United Kingdom
Anti-WT1 is a laboratory product used for research purposes. It is an antibody that recognizes the Wilms' Tumor 1 (WT1) protein, which is a transcription factor involved in various biological processes. This product can be used in techniques such as immunohistochemistry, western blotting, and other applications to detect and study the WT1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
X ray film, by GE Healthcare (1 mentions)
Anti tgf β, by Abcam (1 mentions)
Anti ho 1, by Abcam (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Anti cd68, by Abcam (1 mentions)
Cd206, by Abcam (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ab68908, by Abcam (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Anti zo 1, by Abcam (1 mentions)
Anti lc3b, by Abcam (1 mentions)
Anti connexin 43, by Abcam (1 mentions)
Anti occludin, by Abcam (1 mentions)
Anti claudin 11, by Thermo Fisher Scientific (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
8 protocols using anti wt1
1
Renal Protein Expression Analysis
2
Kidney Precursor Cell Differentiation and SIRT1
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Example 9
Confirmation of Differentiation into Kidney Precursor Cell when SIRT1 Expression is Inhibited
Changes in protein expression of Six2 and WT1 were observed in mouse fetal kidney (conditional knockout mouse: Sirt1 co/co; Hoxb7-Cre (+)) at the first day of birth by specifically inhibiting SIRT1 protein in renal collecting duct cells.
Changes in protein expressions of Six2 and WT1 were measured by immunofluorescence staining. For immunochemical staining, a kidney was extracted from a pregnant mouse or a mouse of the 0th day of birth, was fixed with 4% paraformaldehyde for 6 hours, and then was cut at a thickness of 10 mm to be used for staining. Used antibodies were Anti-Six2 (Abcam; ab68908, Cambridge, UK), Anti-WT1 (Abcam; Cambridge, UK), a Zeiss Z1 microscope and a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany).
As a result, as shown in
3
Immunofluorescence Staining of Schwann Cells
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IF staining of SCs was conducted according to a previous study [66 (link)]. Briefly, SCs were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 1% BSA for 1 h. Then, SCs were incubated with anti-WT1 (1:50, Cat# ab267377, Abcam, Cambridge, MA, USA), anti-Vimentin (1:100, Cat# ab92547, Abcam, Cambridge, MA, USA), anti-ZO-1 (1:100, Cat# ab276131, Abcam, Cambridge, MA, USA), anti-Occludin (1:100, Cat# ab216327, Abcam, Cambridge, MA, USA), anti-Claudin-11 (1:100, Cat# 36-4500, Thermo Fisher Scientific, Waltham, MA, USA), anti-Connexin-43 (1:100, Cat# ab217676, Abcam, Cambridge, MA, USA), anti-N-cadherin (1:100, Cat# ab207608, Abcam, Cambridge, MA, USA), and anti-LC3B (1:200, Cat# ab192890, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then, the SCs were incubated with secondary antibody IgG H&L (1:1000, Cat# ab150073, Abcam, Cambridge, MA, USA) at room temperature for 1 h, followed by the nuclei staining of DAPI (Cat# ab104139, Abcam, Cambridge, MA, USA) for 10 min. The images were captured by a fluorescence microscope (Olympus BX53, Tokyo, Japan).
4
Immunohistochemical Profiling of Kidney Tissue
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Kidney tissues were fixed in a 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), embedded in paraffin, sectioned, and stained with haematoxylin-eosin using standard methods. Immunostaining was carried out using a BlueMap Kit and automated Discovery System (Roche) or manually for immunofluorescence staining.
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
5
Antibody Panel for Multimodal Protein Analysis
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Antibodies used for western blot (WB), immunofluorescence (IF) and chromatin immunoprecipitation (ChIP) were as follows: anti-H3K36me3 (Solarbio no. K003337P, 1:400 for IF), anti-H3K36me2 (Solarbio no. K003336P, 1:400 for IF), anti-H3 (Solarbio no. K107283P, 1:800 for WB), anti-γ-H2A.X (Solarbio no. K006207P, 1:200 for IF, 1:1000 for WB), anti-mCherry (Solarbio no. K200015M, 1:1000 for WB), anti-H4K16ac (Abcam no. ab109463, 1:200 for IF), anti-NSD2 (Abcam no. ab-75359, 1:1000 for WB, 1:200 for IF), anti-PSMA8 (Proteintech no. 4022-1-AP, 1:1000 for WB), anti-GAPDH (Proteintech no. 60004-1-Ig, 1:1000 for WB), anti-PRM2 (Briar Patch Biosciences, Hup2B, 1:200 for IF, 1:800 for WB), anti-WT1 (Abcam no. ab89901, 1:200 for IF), anti-H4K5ac (ABclonal no. A19525, 1:1000 for WB, 1:200 for IF), anti-H4K8ac (ABclonal no. A7258, 1:1000 for WB, 1:200 for IF), anti-EP300 (Abcam no. ab-10485, 1:1000 for WB), anti-H4 (ABclonal no. A1131, 1:1000 for WB) and anti-α-Tubulin (Proteintech no. 66031-1-Ig, 1:1000 for WB).
6
Immunofluorescence Staining Protocol
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Primary antibodies (1/250 dilution): anti-PAX8 (Proteintech, 10336-1-AP), anti-GFP (Abcam, ab13970), anti-mCherry (Abcam, ab213511), anti-WT1 (Abcam, ab89901), anti-acetylated tubulin (Sigma, T7451), anti-Sox17 (R&D, AF1924), anti-FoxA2 (Cell Signaling, 3143), and anti-E-cadherin (Invitrogen, 13-1900) . Secondary antibodies (1/450 dilution): Alexa Fluor (AF) antirabbit 555 (Invitrogen, A31572), AF anti-rabbit 649 (Invitrogen, A21245), AF anti-rabbit 488 (Invitrogen, A21206), AF anti-mouse 649 (Invitrogen, A32787), AF anti-mouse 488 (Invitrogen, A21202), AF anti-goat 568 (Invitrogen, A11057), AF anti-rat 488 (Invitrogen, A21208), antichicken 488 (Sigma, SAB4600031), DAPI/Hoechst 33342 (Thermo Fisher, 62249), Alexa Fluor 488 phalloidin (Lifetech, A12379), and Alexa Fluor 635 phalloidin (Lifetech, A34054).
7
Podocyte Protein Lysate Analysis
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Total protein lysate from NHP podocytes was prepared using RIPA buffer (PIERCE, Cat# 89900). Precast 4–12% gels were used for SDS-PAGE (Invitrogen, Cat# NP0322PK2) and transferred to cellulose membranes using Semi-dry system (BioRad, Cat# 170–3940). Membranes were blocked with 5% milk in TBST for at least 1 h at room temperature or overnight at 4 °C. Primary antibody was incubated for 1 h followed by dye-labeled secondary antibody for 45 min at room temperature. Membranes were then washed three times with TBST before imaging analysis using LI-COR system. Primary antibodies included anti-Adora2b (NHP) (LifeSpan Bioscience, Cat#LS-A680), anti-Adora2b (Rat) (Millipore, Cat#AB1589P), anti-Tubulin (Thermal Fisher, Cat#MA1-19401), anti-Gapdh(Santa Cruz, Cat#SC-25778) anti-Nephrin (Abcam, Cat#ab85379), anti-Synaptopodin (Abcam, Cat#101883), anti-WT-1 (Epitomics, Cat#AC-0115), anti-cleaved Caspase3 (Cell Signaling, Cat#9661S), anti-Caspase-3 (Cell Signaling, #9662), and anti-Actin (SCB, #SC-1616).
8
Immunohistochemical Analysis of Mouse Testes
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Mouse testes were fixed in Bouin’s solution (Sigma-Aldrich, MO, USA) overnight at 4 °C. Paraffin embedding, section preparation, deparaffinization, H&E staining or immunohistochemistry analysis were carried out by using standard histological procedures. For Immunofluorescence, testes were embedded in OCT compound (Sakura Finetek) and frozen in liquid nitrogen. Cryosections (5 μm) were mounted on glass slides and subjected to immunostaining. Primary antibodies were incubated at 4 °C overnight and secondary antibodies were added at room temperature for 2 hr. Nuclei were visualized by staining with DAPI. The following antibodies were used: rabbit polyclonal anti-SYCP3 (ab-15093, Abcam), mouse monoclonal anti-γH2AX Ser-139 (cat. no. 05–636, Millipore), rabbit polyclonal anti-CLGN (12629-1-AP, Protein Tech Group), CREST serum (Immunovision, Springdale, AR), mouse monoclonal anti-α-tubulin (Sigma), rabbit polyclonal anti-MVH (ab13840, Abcam), mouse monoclonal anti-DAZL (LS-C188293, LifeSpan Biosciences), rabbit polyclonal anti-WT1 (Epitomics), mouse anti-PLZF (Calbiochem), rabbit anti-PH3 (Cell Signaling Technology), rabbit anti-CREM (X-12, Santa Cruz), rabbit polyclonal anti-AFAF (a kind gift from Prof. Yi-Xun Liu, Institute of Zoology, Beijing)31 (link)
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