Tissues were fixed in 10% formalin, processed, and embedded in paraffin. Thin (5 μm) sections were cut, dried overnight, and baked at 65°C for 30 min. Antigen retrieval was performed using citrate buffer at pH 6 (Sigma) for 15 min at 100°C. Endogenous peroxidase activity was blocked by incubating the slides in 3% peroxide in methanol for 5 min. All subsequent steps were intermitted by washing in TBS with 0.5% Tween 20. Endogenous biotin was blocked using an avidin/biotin blocking kit (Vector Labs), followed by a 30-min protein-blocking step with serum-free protein block (Life Technologies) at 37°C. Sections were incubated with anti-ETV7 antibody overnight at 4°C. For peptide competition, diluted antibody was incubated with ETV7 peptide at room temperature for 30 min before staining. Biotinylated secondary antibody (Vector Labs) was used at 6 μg/ml for 30 min at room temperature, followed by streptavidin-HRP (Dako) and 3,3′-diaminobenzidine (DAB) chromogen (Dako) following the manufacturer’s protocol. Images were captured using 200× magnification on a Nikon E800 microscope at the Cell Imaging Core Facility of St. Jude Children’s Research Hospital.
Serum free protein block
Manufactured by Thermo Fisher Scientific
The Serum-free protein block is a laboratory reagent used to prevent non-specific binding in immunoassays and other protein detection techniques. It is designed to block unoccupied binding sites on solid supports, reducing background signal and improving the specificity of target protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Citrate buffer, by Merck Group (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine dab chromogen, by Agilent Technologies (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
2 protocols using serum free protein block
1
Immunohistochemical Detection of ETV7
2
Immunohistochemical Detection of Marburg Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Sections from paraffin-embedded blocks containing tracheobronchial lymph node (aerosol study only), inguinal lymph node, liver and spleen were sectioned, deparaffinized, rehydrated, subjected to methanol-hydrogen peroxide block, and rinsed, and antigen retrieval was performed by incubation with TRIS/EDTA Buffer at pH 9.0 for 30 min at 97°C. An immunoperoxidase assay system was used according to the manufacturer's recommendations (Envision System; DAKO lnc., Carpinteria, CA). A serum-free protein block (Life Technologies, Grand Island, NY) plus 5% normal goat serum was applied for 30 minutes followed by staining with a MARV/Angola mouse monoclonal antibody (at a dilution of I: 4000 in 10% normal goat serum) at ambient temperature for 30 min. A horseradish peroxidase-conjugated anti-mouse antibody (Envision kit) was added, and the sections were incubated for 30 min at ambient temperature. All sections were exposed to DAB permanent chromogen (DAKO, Carpinteria, CA) for 5 min, rinsed, counter-stained with hematoxylin, dehydrated, and coverslipped with Permount (Thermo Fisher Scientific, Waltham, MA). The intensity of staining for antigen was semi-quantitatively documented using the following scale: + for 1–25 reactive cells/high power field (hpf); ++ for 25–50 reactive cells/hpf; +++ for > 50 reactive cells/hpf.
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