Staining was performed as described in Yelamanchili et al. 15 . After hydration with a graded alcohol series, tissue was subjected to citrate antigen retrieval and washed with TBS. After blocking in a solution of 1% BSA and 3% NGS in PBS, tissue was incubated with primary antibody at a dilution of 1:500 for overnight at 4 °C. Antibodies used were anti‐GFAP (Dako Z0334, Carpinteria, CA, USA) and anti‐Iba‐1 (Wako 019‐19741, Richmond, VA, USA). On the following day, tissue was washed with TBS and incubated with 3% H202 in PBS for 10 min. After thorough washing, tissue was incubated with anti‐Rabbit secondary labeled with a peroxidase enzyme (ImmPRESS; Vector labs, Burlingame, CA, USA) for 1 h, rinsed, and developed with DAB Plus substrate system (Thermo) for 10 min. Tissue was then washed with TBS, stained with hematoxylin, and dehydrated. Coverslips were mounted with cytoseal (Thermo). Immunostaining was performed on three biological replicates.
Dab plus substrate system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DAB Plus substrate system is a chromogenic detection system used in immunohistochemistry and in situ hybridization applications. It produces a brown insoluble precipitate at the site of the target antigen or nucleic acid, enabling visual identification and localization.
Automatically generated - may contain errors
Lab products found in correlation
Cytoseal, by Thermo Fisher Scientific (2 mentions)
Anti iba1, by Fujifilm (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Hpa008736, by Merck Group (1 mentions)
Hpa001906, by Merck Group (1 mentions)
Immpress hrp anti rabbit igg, by Vector Laboratories (1 mentions)
Citrate buffer ph 6, by Thermo Fisher Scientific (1 mentions)
Primary antibody enhancer, by Thermo Fisher Scientific (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Vslide, by MetaSystems (1 mentions)
Autostainer480s, by Thermo Fisher Scientific (1 mentions)
4 protocols using dab plus substrate system
1
Immunohistochemical Staining Protocol
2
Immunolabeling of TP53, ATRX, and DAXX in ULMS
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TP53, ATRX, and DAXX immunolabeling was performed on FFPE sections of all 19 exome-sequenced ULMSs and on a tissue microarray containing 33 additional tumors. For TP53, immunostaining was performed as previously described [31 (link)]. For ATRX and DAXX, heat-induced antigen retrieval was carried out in a microwave using citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase blocking was followed by overnight incubation with the primary antibody at 4°C (anti-ATRX 1:500 dilution, Sigma-Aldrich catalog# HPA001906; anti-DAXX 1:500 dilution, Sigma-Aldrich catalog# HPA008736). The primary antibody was detected with DAB Plus Substrate System (Thermo Fisher Scientific catalog# TA-060-HDX). Immunohistochemical scoring was assessed by a pathologist (RB). Only nuclear labeling of the proteins was evaluated. The loss of nuclear staining in tumor cells together with retained expression in non-neoplastic cells (endothelial or inflammatory cells) was considered loss of expression. The scoring was done without knowledge of the clinical outcome data.
3
Quantifying Microglial Activation in Brain
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Free-floating cryosections were incubated with 3% H2O2 to quench endogenous peroxidase. After thorough washing sections were blocked with 10% normal goat serum (NGS) +0.3% Triton-X100 in PBS. Tissues were incubated with Iba1 (Wako, Kampenhout; 1:500) in 0.3% Triton-X100 +3% NGS in PBS at 4°C overnight. ImmPRESS HRP Anti-Rabbit IgG (Vector labs, Burlingame) was used as a secondary and DAB Plus substrate system (Thermo, Waltham, MA, USA) was used for developing. Sections were then mounted on gelatin-coated slides, allowed to dry overnight, incubated in xylene for 1 min, and mounted with Cytoseal (Thermo, Waltham, MA, USA). Slides were imaged using a light microscope at 100× magnification. Images were quantified using ImageJ (Schneider et al., 2012 (link)) to calculate the percent area covered by Iba1 staining in the hippocampus and cortex.
4
Immunohistochemical Evaluation of SATB1 and SATB2
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The specificity of SATB1 and SATB2 antibodies was further evaluated in immunohistochemical experiments.
Tissue sections (4 μm) were cut from TMAs containing 18 normal (fallopian tube, cervix, endometrium, placenta, testis, prostate, liver, pancreas, rectum, colon, stomach, duodenum, small intestine, cerebellum, cerebral cortex, skin, skeletal muscle, and tonsil) and 7 cancer (prostate, colorectal, ventricular, renal, liver, lung, and breast) tissues. Prior to immunostaining, the sections were baked at 50 °C overnight and deparaffinized in xylene and graded ethanol. Antigen retrieval was then performed using citrate buffer pH 6 (ThermoFisher Scientific, Waltham, MA, USA) in decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Sections were stained with anti-SATB1rabbit monoclonal antibody (Clone EPR3895, Epitomics, Burlingame, CA, USA) diluted 1:100 or mouse monoclonal antibody against SATB2 (AMAb90679, CL0320, Atlas Antibodies, Stockholm, Sweden) diluted 1:1,000 in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA) using a commercial kit (UltraVision LP HRP polymer®, Primary Antibody Enhancer, Ultra V Block and DAB plus substrate system®, ThermoFisher Scientific, Waltham, MA, USA). Slides were counterstained with hematoxylin and mounted using Pertex.
Slides were examined, and images were taken using an automated system (VSlide, Metasystems).
Tissue sections (4 μm) were cut from TMAs containing 18 normal (fallopian tube, cervix, endometrium, placenta, testis, prostate, liver, pancreas, rectum, colon, stomach, duodenum, small intestine, cerebellum, cerebral cortex, skin, skeletal muscle, and tonsil) and 7 cancer (prostate, colorectal, ventricular, renal, liver, lung, and breast) tissues. Prior to immunostaining, the sections were baked at 50 °C overnight and deparaffinized in xylene and graded ethanol. Antigen retrieval was then performed using citrate buffer pH 6 (ThermoFisher Scientific, Waltham, MA, USA) in decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Sections were stained with anti-SATB1rabbit monoclonal antibody (Clone EPR3895, Epitomics, Burlingame, CA, USA) diluted 1:100 or mouse monoclonal antibody against SATB2 (AMAb90679, CL0320, Atlas Antibodies, Stockholm, Sweden) diluted 1:1,000 in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA) using a commercial kit (UltraVision LP HRP polymer®, Primary Antibody Enhancer, Ultra V Block and DAB plus substrate system®, ThermoFisher Scientific, Waltham, MA, USA). Slides were counterstained with hematoxylin and mounted using Pertex.
Slides were examined, and images were taken using an automated system (VSlide, Metasystems).
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