Vector dab peroxidase substrate kit

Gingival tissue samples with normal gingival apparatus (healthy control) and from periodontal disease patients (deep pockets exceeding 5 mm) were collected from routine dental care, placed in Tissue-Tek^® (OCT compound, Sakura Finetek USA, Inc., Torrance, CA, USA) and snap-frozen in liquid nitrogen. Frozen sections (6–8 μm) were cut with a cryostat and stored at −80 °C until used for immunolocalization studies. To this end, frozen sections were fixed with acetone (−20 °C) for 5 min, rinsed and incubated in normal blocking serum (Vectastain Elite ABC Kit; Vector Laboratories, Inc., Burlingame, CA, USA) in a humidified chamber at room temperature for 30 min. After rinsing, the sections were incubated overnight with the primary antibody against αvβ6 integrin (β6B1; a generous gift from Dr. Dean Sheppard, University of California, San Francisco, CA, USA), followed by an incubation with a biotinylated, horseradish peroxidase-conjugated secondary antibody (Vectastain) for 1 h, incubation with ABC avidin/peroxidase reagent and reaction with Vector DAB Peroxidase Substrate Kit (Vector Laboratories). To stop the reaction, the sections were rinsed with distilled water for 10 min. After counterstaining with hematoxylin, the sections were allowed to air dry, mounted using Vectamount permanent mounting medium (Vector Laboratories), viewed and photographed using a light microscope.

Specimens were obtained from Icahn School of Medicine at Mount Sinai and considered non-human subject research. Tissue Microarray slides were provided by the NCI cancer Diagnosis program (CDP). Other investigators may have received slides from the same blocks. IHC was performed as described before^{62 (link)}. In brief, 5 μm sections from formalin-fixed paraffin-embedded specimens were deparaffinized, incubated for antigen retrieval with Vector Citrate-Based Antigen Unmasking Solution (Vector Laboratories) in microwave for 10 min, and then exposed to 0.3% hydrogen peroxide to block endogenous peroxidase activity. After blocking with Vector Normal Horse Serum (2.5%) for 20 min, sections were incubated at 4 °C overnight with mH2A2 (1:350–1:500) prepared in 0.1% BSA. Slides were subsequently developed using Vector imPRESS Universal Kits anti-mouse/rabbit Ig or anti-goat Ig (Vector Laboratories), Vector DAB Peroxidase Substrate Kit as the chromagen (Vector Laboratories) and Harris Hematoxylin (Sigma) for counterstaining. Slides were then sealed and mounted with Permount (Sigma) and randomized for subsequent blinded review.