Sf98 4

Immunofluorescence staining was performed as previously described with modifications^{12 (link)}. Slides containing brain sections were first post-fixed in 10% neutral buffered formalin (Fisher Scientific, SF98-4) for 5–10 min, then washed three times with PBS. Antigen retrieval was performed using the Antigen Unmasking Solution (Vector Laboratories, H-3300) at 95 °C for 20–60 min in a water bath with gentle shaking (60 rpm). Slides were cooled to room temperature and permeabilized with 0.3% Triton X-100 in PBS, then blocked with 5% normal donkey serum in 0.3% Triton X-100 in PBS. Slides were incubated at 4 °C overnight with the primary antibodies in blocking buffer. The slides were washed three times with 0.3% Triton X-100 in PBS, then incubated with secondary antibodies in blocking buffer at room temperature for two hours. The slides were then washed twice with 0.3% Triton X-100 in PBS, and then once with PBS. Autofluorescence quenching was carried out to reduce background autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, SP-8400) and all slides within one experiment were treated similarly. Slides were counterstained with DAPI and then mounted using VECTASHIELD Vibrance mounting media (Vector Laboratories, H-1700).