Biotinylated anti rat igg secondary antibody

Mouse tissue was diced into 5 × 5-mm sections, immersion-fixed in PBS containing 4% paraformaldehyde for 24 h at 4°C, and embedded in paraffin. Four micrometer sections were mounted on glass slides. Hematoxylin and eosin (H&E) stained liver specimens were evaluated by light microscopy for histopathological scoring by a hepatopathologist (BGP). Steatosis, inflammation, and ballooning were scored based on NAFLD activity score [26 (link)]. Presence of macrophage infiltration was assessed by immunohistochemical staining for F4/80. Paraffin-embedded liver sections were deparaffinized and antigen retrieval using 10 mM sodium citrate buffer was performed. Sections were incubated with primary antibody overnight at 4°C (1:50 dilution, Abd Serotec, Oxford). Subsequently, sections were incubated with a biotinylated anti-rat IgG secondary antibody (Vector Laboratories, Burlingame, CA) and a Vectastain ABC Elite Kit according to manufacturer’s instructions (Vector Labs). Sections were developed with ImmPACT DAB peroxidase substrate (Vector Labs) and counterstained with hematoxylin.

The 5 μm oesophageal paraffin tissue sections were immunostained with antiserum against mouse eosinophil major basic protein (anti-MBP, a kind gift of Drs. James and Nancy Lee, Mayo Clinic, Scottsdale, AZ) as described. ^{48 (link), 49 (link)} In brief, endogenous peroxide in the tissue was quenched with 0.3% hydrogen peroxide in methanol followed by non-specific protein blocking with normal rabbit serum. Tissue sections were then incubated with rat anti-MBP (1:2000) overnight at 4°C, followed by 1:200 dilution of biotinylated anti-rat IgG secondary antibody and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 minutes each. The slides were further developed with nickel diaminobenzidine-cobalt chloride solution to form a black precipitate, and counterstained with nuclear fast red. Negative controls included replacing the primary antibody with normal rabbit serum.