PFA-fixed paraffin sections (4 μm) were mounted on slides, dewaxed with conventional xylene at 60°C and rehydrated with gradient alcohol for 5 min respectively. Slides were washed with PBS and boiled in 10 mM sodium citrate buffer (pH 6.0) for 2 min for antigen retrieval, cooled on the bench top for 30 min, treated with 3% H2O2 at 37°C for 15 min to block endogenous peroxidase activity, and then blocked with 20% goat serum (cat. no. ab138478; Abcam) in PBS-T for 1 h at room temperature. Samples were incubated with primary antibodies (anti-PDPK1; cat. no. ab52893; 1:50; Abcam) overnight at 4°C. Following washing with PBS-T, sections were incubated with an horseradish peroxidase-conjugated secondary anti-rabbit antibody for 20 min at room temperature, rinsed again with PBS-T, and incubated with Lab Vision™ polyvalent detection kit (Thermo Fisher Scientific, Inc.) and DAB [Tiangen Biotech (Beijing) Co., Ltd., Shanghai, China] for 10 min at room temperature. Slides were then observed under an inverted light microscope (magnification, ×400).
3 3 diaminobenzidine (dab)
Manufactured by Tiangen Biotech
Sourced in China
DAB (3,3'-Diaminobenzidine) is a chromogenic substrate used in various immunohistochemical and histological detection techniques. It produces a brown precipitate upon enzymatic reaction, allowing for the visualization of target antigens or molecules in tissue samples or cell preparations.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by GE Healthcare (1 mentions)
Goat anti rabbit igg hrp, by Boster Bio (1 mentions)
Ki 67 antibody, by Boster Bio (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Goat anti human igg hrp, by Abcam (1 mentions)
Elisa microplate reader, by Agilent Technologies (1 mentions)
5 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Analysis of PDPK1
2
Immunoblotting of Protein Samples
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The protein samples from each SDS-PAGE gel were transferred onto PVDF membranes (GE Healthcare) using a semi-dry blotting apparatus (TE77, GE Healthcare) for 2 h at 0.65 mA/cm2. The membrane was then blocked with 5% (w/v) skimmed milk in 50 mM Tris–HCl buffer (pH 7.4) containing 0.05% Tween 20 (TBST) for 2 h at RT. The blocked membrane was then incubated with GD201008-001 hyperimmune serum or “pre-absorbed” serum (1: 200 dilution) for 2 h at RT and washed three times with TBST (10 min per wash). The membrane was incubated with HRP-goat anti rabbit IgG (1:10,000 dilution; Boster) at RT for 1 h, washed three times with TBST, and developed by adding DAB (Tiangen) until the optimum color was obtained. Western blotting was repeated in triplicate.
3
Histological Examination of Intestinal Architecture
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Colon sections were stained with H&E for assessment of intestinal architecture and Periodic acid–Schiff, anti-Reg3g (AP5606c; Abgent) for immunohistochemistry. Slides were washed three times with 0.1% TBS-Tween before incubation with secondary antibodies. Stained slides were washed again in PBS and stained with DAB (TIANGEN) in conjunction with a hematoxylin counterstain (Solarbio). After dehydration, sections were mounted in neutral balsam.
4
Immunohistochemical Analysis of Ki-67 in GC Tumors
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GC tumors from mice were immunostained for Ki-67 with two-step immunohistochemical staining using streptavidin–peroxidase (SP) and diaminobenzidine (DAB). Fresh gastric tumor biopsies collected from nude mice were fixed immediatelsy with 4% paraformaldehyde (Sigma-Aldrich, Irvine, Ayrshire, UK) for 30 min at 30°C. The biopsies were then embedded in paraffin, sectioned (4 μm thick) onto slides, and rehydrated in xylene. Antigen retrieval was performed in 20 mmol/L sodium citrate-repairing solution (pH 6.0) at 95°C for 15 min. The tissue sections were then incubated in 3% H2O2 solution for 10 min to block endogenous peroxidase activity and incubated with 1:50 diluted Ki-67 antibody (Boster Biological Technology, Ltd., Wuhan, P.R. China) at 4°C overnight. PBS solution as a substitute for primary antibody was used as the negative control. After rinsing three times with PBS, the tissue sections were further incubated with the appropriate secondary antibodies at 37°C for 30 min followed by exposure to SP for another 30 min. Subsequently, the tissue section was stained with DAB (Tiangen Biotechnology, Beijing, P.R. China), counterstained with hematoxylin, dehydrated, and sealed.
5
Indirect ELISA for SLE Autoantibody Detection
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Serum samples were collected from all SLE patients and HCs. 100 µl EP (10 µg/mL) was coated in each microplate. 1:50 diluted serum sample was used as primary antibody and sealed with 5% skim milk, while 1:10,000 diluent goat anti-human IgG-HRP (Abcam, USA) was applied as secondary antibody to carry out indirect ELISA. Post being incubated with DAB (TIANGEN, China), OD was subsequently measured at 450 nm by using a Bio-Tek ELISA microplate reader. All samples were independently analyzed in triplicate, and the mean value of OD was used for analysis in this study.
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