Slides were deparaffinized and hydrated to water then stained on the Thermo/Labvision Auto-stainer. Sections were incubated in Endogenous Peroxidase Blocker (DAKO, S2003) for 10 min. Followed by proteolytic digestion with Proteinase-K (DAKO S3020) for 1 min. Additional blocking was done with Background Buster (Innovex, NB306) for 30 min. Slides were then incubated with the primary antibody, a polyclonal rabbit anti-ASO antibody (8 (link)) at a dilution of 1:10 000 (diluted in 2% bovine serum albumin, 5% donkey serum) for 1 h. A secondary antibody Donkey anti Rabbit HRP (Jackson, 711-036-152) was applied for 30 min. Liquid DAB+ Substrate Chromogen System (Dako, K3468) was used to visualize the ASO antibody. Slides were counterstained with Hematoxylin for 30 s then dehydrated, cleared, and coverslipped with Micromount (Leica, 3801731).
Endogenous peroxidase blocker
Manufactured by Agilent Technologies
Sourced in United Kingdom
Endogenous peroxidase blocker is a laboratory reagent used to inhibit endogenous peroxidase activity in biological samples. It is a key component in various immunohistochemical and immunocytochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Agilent Technologies (2 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Donkey anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Micromount, by Leica (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Agilent Technologies (1 mentions)
Target retrieval solution ph 6, by Agilent Technologies (1 mentions)
Vs200 slide scanner, by Olympus (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
5 protocols using endogenous peroxidase blocker
1
Immunohistochemical Localization of ASO
2
Mucin-like Protocadherin Expression in Colorectal Cancer
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Immunohistochemical staining was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections of CRCs and matched normal mucosa from 14 patients, together with 10 colorectal adenomas using the Envision Detection System (DAKO). Sections were deparaffinized with xylene and rehydrated in a series of ethanol solutions decreasing in concentration. Heat-induced epitope retrieval was carried out in a Target Retrieval Solution pH 6 (DAKO) in a 96°C water bath for 20 minutes. After cooling, retrieval solutions were kept at room temperature for 25 minutes and the slides were treated for 5 minutes with an Endogenous Peroxidase Blocker (DAKO). Slides were then incubated with monoclonal antibody against mucin-like protocadherin (PA5-32704, Pierce/Thermo Scientific) at 1 : 300 dilution for 30 minutes at room temperature and subsequently labelled with the Envision Detection System (DAKO). The colour reaction product was developed with 3,3′-diaminobenzidine, tetrahydrochloride (DAB) (DAKO) as substrate and nuclear contrast was achieved with hematoxylin counterstaining. Staining intensity was assessed by a four-grade scale: 0: lack of expression; 1: weak expression; 2: moderate expression; and 3: strong expression. The stained tissue slides were examined by the pathologist blinded to the qRT-PCR results.
3
Immunohistochemical Analysis of Liver Tissue
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Immunohistochemistry was performed as previously described.37 (link) Briefly, liver tissue sections (5 μm) were deparaffinized in xylene and rehydrated in serial dilutions of ethanol. After deparaffinization, antigen retrieval was performed at boiling temperature in citrate buffer (10 mM, pH 6.0) for 20 min for CD45 or in presence of proteinase K (Dako, Jena, Germany) for 5 min at room temperature (RT) for F4/80 antigen. Slides were incubated with endogenous peroxidase blocker (Dako) for 20 min at RT, followed by blocking of endogenous biotin (Abcam, Cambridge, UK). Next, sections were incubated with primary antibodies at 4 °C overnight (Supplementary Table S2 ). On the next day, slides were washed with phosphate-buffered saline (PBS) for 5 min and incubated with secondary antibodies for 1 h at RT (Supplementary Table S3 ). Brown color staining was developed with diaminobenzidine (DAB; Sigma-Aldrich) and nuclei were counterstained with hematoxylin (Sigma-Aldrich). Stained slides were scanned by a VS200 slide scanner (Olympus, Tokyo, Japan). Percentage of positive stained area was calculated as described before. Liver tissues incubated with PBS instead of primary antibody served as negative control.
4
Immunohistochemical Analysis of TSPAN1
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Tissues were stored in formalin zinc fixative for 16–24 h and then transferred to 70% ethanol solution for paraffin embedding. Slides were blocked with endogenous peroxidase blocker (Dako) or Bloxall endogenous peroxidase and alkaline phosphatase blocker (Vector Laboratories) according to product instructions and then incubated with horse serum for 1 h at room temperature (Vector Laboratories). Primary Abs were incubated overnight at 4°C on slides. The next day, slides were treated with the appropriate antispecies enzyme‐labeled secondary Ab for 30 min at room temperature. All slides were made using DAB Peroxidase Substrate Kit (Vector Laboratories) unless otherwise noted. The TSPAN1 immunostaining was assessed using a modified semiquantitative immunoreactive score system, incorporating clinical data. The scoring criteria for category A (immunostaining intensity) was graded as: 0, negative; 1, weak; 2, moderate; and 3, strong. Category B (percentage of immunoreactive cells) was graded as: 1, 0%–25% cells; 2, 26%–50% cells; 3, 51%–75% cells; and 4, 76%–100% cells. The final scores were calculated by multiplying the scores of categories A and B for each section, resulting in scores ranging from 0 to 12. A TUNEL kit was used for apoptosis measurement according to the manufacturer's protocol.
5
Comprehensive Immunohistochemical Profiling of FFPE Tumor Samples
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Immunohistochemical staining was performed on 4-µm FFPE tumor sections using the EnVision FLEX+, Mouse, High-pH Detection System (Dako, Glostrup, Denmark). Sections were deparaffinized with xylene and rehydrated in ethanol solutions. Heat-induced epitope retrieval was carried out in Target Retrieval Solution (Dako) for 20 min at 96 °C. After cooling, the slides were treated for 5 min with an endogenous peroxidase blocker (Dako), incubated with monoclonal antibody against androgen receptor (D6F11, Cell Signaling), BRG1, INI1, BAF155, BAF170, BAF250a and BRM (P680, D9C2, D7F8S, D8O9V, D2A8U, D9E8B, Cell Signaling) for 30 min at room temperature, and then labeled with the EnVision FLEX+, Mouse, High-pH Detection System (Dako). The color reaction product was developed with 3,3′-diaminobenzidine tetrahydrochloride (Dako) as a substrate, and nuclear contrast was achieved with hematoxylin counterstaining.
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