Dapi containing mounting solution

Fixation was carried out with ice-cold 4% paraformaldehyde (2 h for OHC; 30 min for cells), followed by permeabilization when necessary with 0.1% Triton X-100 on ice (1 h for OHC; 10 min for cells) and blocking in 3% BSA (2 h for OHC; 30 min for cells). Incubation with primary antibodies was carried out in a 3% BSA solution (24 h for OHC; overnight for cells) at 4 °C. Primary antibodies used were: chicken anti-integrin α-M (integrin; 1:80, Aves Labs MAC, Tigard, OR, USA), mouse anti-GFAP (1:300, Cell Signaling #3670, Danvers, MA, USA), rabbit anti-BDNF (1:100; S. Cruz), mouse anti-MAP2 (1:100; #05-346, Merck-Millipore); mouse anti-SYP (1:150; S. Cruz). After washing, cells were incubated with secondary antibodies RT (2 h for OHC; 45 min for cells), washed and mounted with regular or DAPI-containing mounting solution (both from Sigma). Secondary antibodies used were: PE-anti-chicken (1:200; S. Cruz sc-3730), Alexa-Fluor 546-anti-mouse (1:300; Invitrogen A10036), Alexa-Fluor 488-anti-rabbit (1:300; Invitrogen A11088). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany).