Donkey anti rat igg alexa 488 staining

Fixed and cryoprotected GBM tissues were sectioned at 5 μm on positive-coated slides and rehydrated for 15 min in phosphate-buffered saline (PBS), pH 7.4. The sections were then post-fixed in 4% paraformaldehyde for 15 min and rinsed in PBS. Non-specific background was blocked by incubation in blocking solution (5% goat serum, 5% donkey serum, 0.3% Triton X-100 in PBS, pH7.4) for 1 h at room temperature. The sections were incubated with anti-SOX2 rat monoclonal antibody (ThermoFisher #14–9811-82, 1:200 dilution) overnight at (+4°C) followed by donkey anti-rat IgG Alexa 488 staining (ThermoFisher #A-21208, 1:1,000 dilution). Labeling of FOXO3 was performed subsequently by using anti-FOXO3 (D19A7) rabbit monoclonal antibody (Cell Signaling Technologies, #12829, 1:200 dilution) 1 h at room temperature. To enhance FOXO3 staining, VectaFluor Excel Amplified AntiRabbit IgG, DyLight 594 Antibody Kit was used according to the manufacturer’s protocol. The slides were mounted with ProLong Glass Antifade Mountant with NucBlue Stain (ThermoFisher #P36981) and cured overnight at room temperature.