Rabbit anti involucrin

Formalin fixed, paraffin embedded and sectioned (5 μm) skin tissues were used for histological analysis (H&E staining) and immunohistological detection of Mmp-9 and involucrin expression. For immunohistochemistry skin sections were deparaffinized in xylene (Sigma-Aldrich), treated with 0.75% glycine (Sigma-Aldrich) for 30 min to block free aldehyde groups and with 3% H₂O₂ (Sigma-Aldrich) for 5 min to block endogenous peroxidases. Slides were blocked in 10% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Primary antibodies: rabbit anti-involucrin (1:1500, Covance) or rabbit anti-Mmp-9 (1:100, Millipore) were applied (o/n; 4 °C). Antibody binding was detected with the ABC complex (Vectastain ABC kit from Vector Laboratories). Peroxidase activity was revealed using 3,3′-diaminobenzidine tetrahydrochloride (Sigma- Aldrich) as a substrate. Two types of controls were performed: (a) the primary antibody was omitted during the procedure; (b) the primary antibody was substituted with nonspecific immunoglobulin G (IgG) during the immunostaining procedure. Tissue sections were mounted undercoverslips with DPX (Merck; Kenilworth, NJ, USA).

The immunofluorescence procedure was based on a published protocols [12 (link), 57 (link)] with modifications. Skin tissues for immunofluorescent detection of involucrin were fixed for 2 h in 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (PB). Fixed tissues were stored in 18% sucrose (Sigma-Aldrich) in PB with 0.01% sodium azide (Sigma-Aldrich). Cryostat sections (8 μm) were blocked in 10% normal horse serum (Vector Laboratories) in 0,1% BSA in PB for 1 h at RT and incubated with rabbit anti-involucrin (1:1500; Covance) antibodies o/n at 4 °C. Tissue sections were subsequently incubated with Alexa Fluor 594 (Life Technologies) for 1 h at RT. Slides were mounted under coverslips using ProLong® Gold Antifade Mountant with DAPI (Life Technologies).
Sections were visualized and photographed with an Olympus microscope (BX43) equipped with an Olympus digital camera (XC50) and analyzed with CellSens Dimension 1 Software (Olympus Soft Imaging Solutions GmbH).