Mouse anti map2

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).

Immunostaining for MBP and MAP-2 was employed to study the integrity of the myelin sheath and the dendrites around the hematoma post-ICH induction. Animals from each group were sacrificed at day 7 or 14 post-ICH induction (n = 5 or 6/group at each time point). Briefly, rats were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS) and 4% buffered paraformaldehyde (pH 7.4). Rats’ brains were rapidly removed, post fixed in 4% formalin for 24 hours, and then cryoprotected in 30% sucrose for 3–4 days at 4 °C. Next, brains were embedded in an optimal cutting temperature compound (SAKURA, Japan) and cryosectioned through the coronal direction at 18-mm thickness. Sections were then permeabilized with 0.3% Triton X-100 in PBS for 30 min, blocked with 5% bovine serum for 1 hour, and incubated at 4 °C overnight with primary antibodies: goat anti-MBP (1:200, Santa Cruz Biotechnology) and mouse anti-MAP-2 (1:200, Merck Millipore). The specimens were incubated with appropriate secondary antibodies for 3 hours at 37 °C. Immunofluorescence was examined under a fluorescent microscope (LSM780, Zeiss, Jena, Germany), and the number of axons per field was counted using Image-pro plus software (Media Cybernetics, Rockville, MD, US).