Biotinylated goat anti rabbit igg h l secondary antibody

The cells seeded in coverslips were fixed in 4% formaldehyde/PBS for 15 min at RT, washed twice with PBS and permeabilized with acetone for 5 min at 4 °C. They were washed again and blocked with 1% BSA in PBST (PBS +0.1% Tween 20) for 30 min at RT. After two more washes with PBS, the preparations were incubated with anti-Cytokeratin 18 antibody, rabbit monoclonal (clone E431-1, Millipore) in a dilution 1:50 in 1% BSA/PBST overnight followed by the incubation of biotinylated goat anti-rabbit IgG (H + L) secondary antibody (Vector Laboratories, Burlingame, CA) at a 1:100 dilution for one hour. After four washes with PBS, the cells were incubated with peroxidase-conjugated avidin (Invitrogen) for one hour in humid chamber and stained with three, 3′-diaminobezidine (DAB) peroxidase substrate kit (Vector Laboratories) for 10 min at RT and, after washing, counterstained with hematoxylin.

After euthanasia, kidneys were flushed with saline, immersion fixed in 4% paraformaldehyde and embedded in paraffin and sectioned (4 µm) (Histology Platform, Monash University). Antigen retrieval was performed by treating sections with citrate buffer (DAKO) for 30 min at 98°C (Monash Histology Platform). Endogenous peroxidase activity was quenched (0.3% hydrogen peroxide) and non-specific binding blocked with 10% goat serum (1:10 dilution, Vector Laboratories). Slides were incubated in a rabbit anti-tyrosine hydroxylase primary antibody (1:800, AB152, Merck Millipore) at 4°C overnight. The next day, sections were incubated in a biotinylated goat anti-rabbit IgG (H+L) secondary antibody (1:200 dilution, Vector Laboratories) and then in the Avidin-Biotin complex (ABC kit, Vector laboratories). Color was developed with 3,3′-diaminobenzidine (DAB kit, Vector laboratories) and then counterstained with haematoxylin, cover slipped and imaged. Quantification of tyrosine hydroxylase was performed as previously detailed [23 (link)].