Dm4000 b microscope

The immunohistochemical staining procedure started with the incubation in 1% hydrogen peroxidase (in PBS:Methanol 1:1) followed by 3 times of 15 min washing with PBST and 30 min incubation in 7% normal horse serum in 0.02% PBST (blocking solution). Sections were incubated ON with the desired primary antibody in blocking solution at 4 °C. The next day, sections were washed 3 times for 15 min with 0.02% PBST, followed by incubation with the secondary biotinylated antibody for 30 min (Vectastain Elite ABC Kit, Vector laboratories). After three 15 min washing steps with 0.02% PBST and incubation for 30 min in an avidin-biotin complex solution (Vectastain Elite ABC Kit), sections were washed two times for 15 min in 0.02% PBST and twice for 15 min in PBS before stained with 3,3′-diaminobenzidine tetra hydrochloride (DAB) solution (DAB substrate Kit for Peroxidase, Vector laboratories, Burlingame, USA). Staining reactions were terminated upon visual inspection by adding and washing with water. Sections were finally air-dried and mounted with Mowiol. Bright field colored images were captured with an automated Leica DM4000 B microscope coupled to a MBF CX9000 camera (MBF Bioscience) and displayed with PictureFrame^™ software.