Immunofluorescent Labeling of PHAL

One series of sections was stained for PHAL using the free-floating method for immunofluorescence. Briefly, sections were transferred to a blocking solution containing normal donkey serum (Vector Laboratories) and Triton X-100 (VWR) for 1 hour. Following three 5-minute rinses, sections were incubated in a KPBS solution comprised of donkey serum, Triton, and a 1:1000 concentration of rabbit anti-PHAL antibody for 48–72 hours at 4°C (Vector Laboratories, #AS-2300; see our previous work^{53 (link)} for validation of this antibody). Sections were rinsed three times in KPBS and then soaked for 3 hours in the secondary antibody solution, which contained donkey serum, Triton, and a 1:500 concentration of anti-rabbit IgG conjugated with Alexa Fluor® 488 or 647 (Invitrogen, 488: #A-21206, 647: #A-31573). Following three KBS rinses, the sections were counterstained with a fluorescent Nissl stain, NeuroTrace® 435/455 (NT; 1:500; Invitrogen, #N21479). The sections were then mounted and coverslipped using 65% glycerol.