4.9. Immunohistochemistry

Immunohistochemical detection of β -galactosidase was performed on cryosections of kidney and lung tissue. The tissue sections were first fixed in 4% paraformaldehyde. All washes were done with phosphate-buffered saline (PBS). The samples were treated for 10 min with 3% H₂O₂ in methanol and for 2 hours with 0.4% pepsin in 0.01M HCl at 37°C. The blocking of nonspecific binding was done by incubating the samples for 20 min at room temperature with 1% bovine serum albumin in PBS followed by the addition of 1:50 – 1:100 dilution of rabbit polyclonal anti-β -galactosidase (Rockland, USA) as a primary antibody. After overnight incubation at +4°C, the peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch laboratories inc, USA) was used as a secondary antibody for one hour at room temperature (dilution 1:600). Peroxidase activity was revealed by 3,3-diaminobenzidine tetrahydrochloride, DAB (Amresco, USA) or 3-amino-9-ethylcarbazole, AEC (Zymed, USA). The sections were counterstained with hematoxylin.

The sections were analyzed visually on a photomicroscope for the presence of the β -galactosidase protein seen as brownish red coloured cells.