Biotinylated goat anti rat igg secondary antibody

5-μm esophageal paraffin tissue sections of mouse esophagus were immunostained with antiserum against mouse eosinophil major basic protein (anti-MBP) as per the method described previously. ^{25 (link)–27 (link)} In brief, endogenous peroxidase. In. the. tissues was quenched with 0.3% hydrogen peroxide in methanol followed by non-specific protein blocking using 3 % normal goat serum. Tissue sections were then incubated with rat anti-MBP (1:6000 dilution) overnight at 4°C (purchased from Mayo Clinic, Scottsdale, AZ, USA), followed by a 1:250 dilution of biotinylated goat anti-rat IgG secondary antibody and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 minutes each. 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 to control for endogenous biotin and peroxidase activity. Eosinophils were quantified by counting the anti-MBP positive-stained cells in each tissue section with the assistance of digital morphometry using the Metamorph Imaging System (Universal Imaging Corp, West Chester, PA) and expressed as eosinophils/mm² tissue area as described earlier. ^{28 (link), 29 (link)}

Paraffin embedded lung tissue sections (5µm) were immunostained with antiserum against mouse eosinophil major basic protein (anti-MBP) as described previously. ^{33 (link), 35 (link)–37 (link)} In brief, endogenous peroxidase in the tissues was quenched with 0.3% hydrogen peroxide in methanol followed by non-specific protein blocking using 3 % normal goat serum. Tissue sections were then incubated with rat anti-MBP antibody (1:6000 dilution) overnight at 4°C (kindly provided by Drs. James and Nancy Lee, Mayo Clinic, Scottsdale, AZ, USA), followed by a 1:250 dilution of biotinylated goat anti-rat IgG secondary antibody and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 min each. 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 to control for endogenous biotin and peroxidase activity.