Pk 6105

Mammary glands were fixed in 10% Neutral buffered formalin for 16–18 hr at 4 °C and paraffin embedded. 3 µM paraffin section slides were first de-paraffinized with xylene, and then rehydrated in descending grade of alcohol (100%, 95%, 70%, and 50%). Samples were washed briefly with PBS before transferring to boiling antigen-unmasking solution (Vector Labs, H-3300) for 20 min. Endogenous peroxidase was blocked by pre-incubating slides in 3% hydrogen peroxide for 10 min followed by 10% normal goat serum in PBS for 1 hr blocking at room temperature. Primary antibody (anti-NELF-B/COBRA1, 1:50) was added and incubated overnight at 4 °C. For detection with primary antibody using the immune enzymatic method, the ABC peroxidase detection system (Vector Labs, PK-6105) was used with 3,3′-diaminobenzidine (DAB) as substrate (Vector Labs, SK-4105) according to manufacturer’s instruction.

After deparaffinization and rehydration, antigens were retrieved using proteinase K (10 ng/ml in PBS) at room temperature for 10 min. Sections were rinsed three times with PBS and incubated with DAKO dual endogenous enzyme blocking reagent (DAKO S2003) at room temperature for 30 min. The slides were rinsed three times with 0.1% Tween-20 in PBS (PBST) and then blocked with 2% normal goat serum at room temperature for 30 min. Next, slides were incubated at 4 °C overnight with antibodies raised against SOX-9 (Abcam, ab 26414, 1:200), Collagen-II (Thermo Scientific, MS235-P, 1:200), MMP13 (Thermo-fisher, MS-825-P, 1:100), or the Aggrecan C-terminal neoepitope NITEGE (MD Bioproducts, 1042003, 1:200). Sections were rinsed three times with PBST and incubated at room temperature for two hours with the appropriate secondary antibody (Vector Laboratories, PK-6105). Color reaction was performed using Vector ImmPact DAB kit (Vector Laboratories, SK-4105). Relative staining intensity and cell quantity (%) were quantified using Image J software (Image J V1.8.0).