To visualize the presence of neurofibrillars tangles, we used human PHF-tau Mab (clone AT100) primer antibody at 1:800 dilution in PBS (pH 7.4) for immunostaining.
After quenching of endogenous peroxidase activity and a blocking step, the sections were incubated overnight at 4 °C with the primary antibody in the presence of 20% goat serum and Triton X-100 0.2%. On the following day, the sections were washed in PBS and incubated 1 h at room temperature with the second biotinylated goat anti-rat antibody (Vector Laboratories, Burlingame, CA, USA, 1:400). The next step was a 1-hour incubation with avidine-biotin complex (Vectastain Elit ABC Kit, Vector Laboratories, Burlingame, CA, USA; 1:400) and detection with nickel-enhanced 3,3′-diaminobenzidine. After immunostaining and washing, all sections were mounted on gelatin-coated slides, air-dried, dehydrated and coverslipped with DPX, a synthetic resin mounting media for histology (Fluka BioChemika, Buchs, Switzerland). Digital photographs were taken by a digital slide scanner (Mirax Midi, Carl Zeiss, Hungary); for the analysis, we used the Histoquant program (3DHistech, Budapest, Hungary).
After quenching of endogenous peroxidase activity and a blocking step, the sections were incubated overnight at 4 °C with the primary antibody in the presence of 20% goat serum and Triton X-100 0.2%. On the following day, the sections were washed in PBS and incubated 1 h at room temperature with the second biotinylated goat anti-rat antibody (Vector Laboratories, Burlingame, CA, USA, 1:400). The next step was a 1-hour incubation with avidine-biotin complex (Vectastain Elit ABC Kit, Vector Laboratories, Burlingame, CA, USA; 1:400) and detection with nickel-enhanced 3,3′-diaminobenzidine. After immunostaining and washing, all sections were mounted on gelatin-coated slides, air-dried, dehydrated and coverslipped with DPX, a synthetic resin mounting media for histology (Fluka BioChemika, Buchs, Switzerland). Digital photographs were taken by a digital slide scanner (Mirax Midi, Carl Zeiss, Hungary); for the analysis, we used the Histoquant program (3DHistech, Budapest, Hungary).