Goat anti mbp

After blocking with 5% bovine serum albumin in PBS/0.3% Triton X-100 for 1 h, free-floating coronal sections (25 μm) were incubated with primary antibodies at 4°C overnight followed by the appropriate secondary antibodies for 1 h at room temperature. The following primary antibodies were used: rabbit anti-Iba1 (1:1000, Wako Diagnostics), goat anti-MBP (1:500, Santa Cruz Biotechnology), rabbit anti-NF200 (1:500, Abcam), and rabbit anti-GFAP (1:500, Dako). Images were acquired via EVOS FL fluorescent microscope (Invitrogen).

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).

Cultured cells or tissues were solubilized in sample buffer, and the protein concentration of each sample was determined using a Beyotime protein assay kit with bovine serum albumin as the standard. Immunoblot analysis (30 μg of protein per lane) was conducted using a 4–10% SDS gradient polyacrylamide gel followed by a standard blotting procedure. Primary antibodies that selectively recognize goat anti-MBP (1 : 250; Santa Cruz; Cat: sc-13914, USA) and actin (1 : 2000, Santa Cruz, USA) were used. Images of the blots were analyzed using ImageJ software (NIH, USA).

Free floating sections were blocked with 1% bovine serum albumin (BSA) and 0.5% Triton-X 100 for 1 h at room temperature and then sequentially incubated with primary antibodies overnight at 4°C and the fluorescent-dye-conjugated secondary antibodies for 2 h at room temperature. Primary antibodies included: rabbit anti-NG2 (1:200, Millipore, Temecula, CA, United States), goat anti-MBP (1:500, Santa Cruz Biotechnology, Dallas, TX, United States), mouse anti-CC1 (1:500, EMD Millipore, Darmstadt, Germany), rabbit anti-HDAC1 (1:200, Invitrogen, Cat: # PA1-860), goat anti-GFAP (1:500, Santa Cruz, Cat: sc-6170), rabbit anti-Iba1 (1:600, Wako, Cat: 019-19741), mouse anti-Vimentin (1:500, Thermo, Cat. # MS-129-R7), Rat anti-CD68 (1:500, abcam, ab53444). Appropriate Alexa Fluor-conjugated secondary antibodies included: donkey anti-mouse, rabbit anti-goat, donkey anti-rat, donkey anti-rabbit IgG (1:1,000, Life Technologies, Carlsbad, CA, United States). DAPI (1:1,000, Invitrogen).