Frozen tumor sections were sequentially fixed with cold acetone, acetone plus chloroform (1:1), and acetone. Paraffin-embedded sections were deparaffinized and heated in antigen retrieval buffer. Tissue sections were blocked with 3% H2O2 in distilled water for 20 min and then in blocking buffer (5% normal horse serum and 1% normal goat serum in PBS). Slides were incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour at room temperature. For immunohistochemistry staining, the secondary antibody was biotin conjugated, the sections were treated with ABC (avidin-biotin complex) reagent (Vector Labs), and the nuclei were counterstained with hematoxylin (Sigma-Aldrich). Tumor sections were mounted with Cytoseal mounting medium (Life Technologies). Quantifications of immunohistochemistry images were assessed by examining three randomly selected low-power fields per slide. For immunofluorescence staining, tumor sections were mounted in an antifade fluorescence mounting medium with 4′,6-diamidino-2-phenylindole. Slides were visualized under a Nikon Eclipse Ti fluorescence microscope.
Abc avidin biotin complex reagent
Manufactured by Vector Laboratories
Sourced in United States
The ABC (avidin-biotin complex) reagent is a tool used in various biomolecular techniques. It consists of avidin, a protein that binds strongly to biotin, a small vitamin molecule. The ABC reagent allows for the detection and visualization of biotinylated targets, such as proteins, nucleic acids, or other biomolecules, in research and diagnostic applications.
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Lab products found in correlation
2 protocols using abc avidin biotin complex reagent
1
Immunohistochemistry and Immunofluorescence Staining of Frozen and Paraffin-Embedded Tumor Sections
2
Immunohistochemistry of eNOS in Mouse Brains
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Brains from 4-week-old A20 HT and KO as well as WT mice were processed for immunohistochemistry (IHC), as previously described (35 (link)). In brief, coronal slices were zinc-fixed (BD Pharmigen, San Diego, CA, USA) for 48 h before paraffin embedding and sectioning (6 μm). Sections were deparaffinized, rehydrated, fixed in cold acetone:formalin 95:5 (vol/vol) for 3 min, and then incubated for 1 h with horse serum (7% in PBS) prior to overnight incubation at 4°C with a polyclonal rabbit-anti-eNOS antibody (Abcam Inc., Cambridge, MA, USA). Sections were then treated with H2O2 1:100 in PBS for 10 min, incubated with the appropriate secondary IgG antibodies followed by ABC (avidin-biotin complex) reagent (Vector Laboratories, Burlingame, CA, USA) and the ImmPACT 3,3′-diaminobenzidine tetrahydrochloride (DAB) peroxidase substrate (Vector Laboratories, Burlingame, CA, USA).
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