Alexa 594 conjugated donkey anti rabbit igg secondary antibody

Fresh frozen coronal sections were obtained from P2 and P8 wild-type and TRPV4-KO mice using a cryostat. After the sections were air-dried, they were fixed with Bouin's fixative for 15 min at 4 °C and incubated with a rabbit anti-TRPV4 antibody (1:400) (Alomone Lab, Jerusalem, Israel) in phosphate-buffered saline (PBS) containing 5% normal donkey serum and 0.3% Triton X-100 overnight at 4 °C followed by an Alexa 594-conjugated donkey anti-rabbit IgG secondary antibody (Invitrogen). Specificity was confirmed by signal ablation in the TRPV4-KO mice. To confirm TRPV4 expression in basal cells, some fixed sections were reacted with rabbit anti-TRPV4 and guinea pig anti-keratin pan (PROGEN, Heidelberg, Germany) antibodies in PBS containing 5% normal goat serum and 0.3% Triton X-100 overnight at 4 °C, followed by incubation with Alexa 594-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-guinea pig IgG secondary antibodies (Invitrogen).