Formalin fixed ovarian tissue was paraffin embedded, 5 μM sections were cut and mounted on SuperFrost plus microscopic slides. Following deparaffinization and rehydration, slides were treated with 3% (v/v) hydrogen peroxide for 10 mins to inhibit endogenous peroxide activities. Tissues were then blocked in 5% bovine serum albumin with 0.02% sodium azide for 10 min and incubated with primary antibodies overnight at 4°c. Following 1x PBS rinse, biotinylated secondary antibodies from respective species were applied for a 2-h incubation at room temperature (1:100 dilution, Sigma) with horseradish peroxidase (Extravidin, 1:50 dilution, Sigma) followed by a brief incubation with diaminobenzidine tetrahydrochloride (Sigma). Tissues were counterstained with Carazzi’s hematoxylin for 1 min, dehydrated and mounted with Permount (Sigma).
For immunofluorescence, after rehydration and blocking with 5% BSA, sections were simultaneously stained overnight with anti-CD31 and anti-NG-2. Sections were stained with secondary antibodies against anti-CD31 (Alexa Fluor®594 nm, red, 1:100) and α-SMA (Alexa Fluor®488 nm, green, 1:100) for 1 h at room temperature. Images obtained under both 594 nm and 488 nm channels using a Fluorescent microscope (Olympus) and Metamorph Imaging software (Burlingame CA).
For immunofluorescence, after rehydration and blocking with 5% BSA, sections were simultaneously stained overnight with anti-CD31 and anti-NG-2. Sections were stained with secondary antibodies against anti-CD31 (Alexa Fluor®594 nm, red, 1:100) and α-SMA (Alexa Fluor®488 nm, green, 1:100) for 1 h at room temperature. Images obtained under both 594 nm and 488 nm channels using a Fluorescent microscope (Olympus) and Metamorph Imaging software (Burlingame CA).