Rat anti mbp

Mice were euthanized and perfused with 15 ml ice-cold phosphate buffer saline (PBS), followed by 20 ml 4% ice-cold paraformaldehyde in PBS. Nerves and brains were harvested; fixed in 4% ice-cold paraformaldehyde overnight; cryoprotected with 10, 20, and 30% ice-cold sucrose in PBS for 4 h, 4 h, and overnight, respectively; and frozen in OCT (Sakura Inc). Ten Micrometre nerve sections and 40 μm brain sections were made for staining. Sections were blocked with 5% normal donkey serum plus 0.3% Triton® X-100 in PBS at room temperature for 1 h, incubated with primary antibody in block at 4 °C overnight. Antibodies and dilutions were: rabbit anti-p75NTR (Cell Signaling Technology, 8238, 1:2000), rat anti-MBP (Biorad, MCA409S, 1:100), goat anti-Gfap (Abcam, ab53554, 1:1000), mouse anti-NeuN (Millipore, mab377, 1:200), goat anti-Iba1 (Abcam, ab5076, 1:200), and mouse anti-CNPase (Millipore, mab326, 1:200). Following incubation with primary antibody, sections were washed three times with PBS, incubated with Alexa fluorophore-conjugated secondary antibodies (1:500, Invitrogen) in block at room temperature for 1 h, washed two times with PBS, incubated with 300 nM DAPI (Sigma) at room temperature for 10 m, washed two times with PBS, and mounted for confocal imaging (Perkin Elmer). TdTomato was strong endogenously and did not require an anti-RFP antibody.

The antibodies used were as follows: rat anti-Ki67 (#14-5698-82, Thermo Fisher Scientific, 1:500), rabbit anti-Olig2 (#AB9610, Millipore, 1:400), rat anti-MBP (#MCA409S, Bio-Rad Laboratories, 1:500), mouse anti-CC1 (#ab16794, Abcam, 1:200), goat anti-PDGFRα (#AF1062, R&D Systems, 1:100), rabbit anti-PDGFRα (#3164 S, Cell Signaling Technology, 1:1000), mouse anti-P65 (#6956 S, Cell Signaling Technology, 1:1000), mouse anti-p-P65 (#3036 S, Cell Signaling Technology, 1:1000), mouse anti-Actin (#M1210-2, HUABIO, 1:5000), mouse anti-Tubulin (#M1305-2, HUABIO, 1:5000), mouse anti-RIPK1 (#610458, BD, 1:1000), rabbit anti-p-RIPK1 (#65746, Cell Signaling Technology, 1:1000). Secondary antibodies: Cy^TM3 affinipure donkey anti-rat IgG (H + L) (#712-165-153, 1:400), Cy^TM3 affinipure donkey anti-mouse IgG (H + L) (#715-165-151, 1:400), Alexa Fluor 488 affinipure donkey anti-rabbit IgG (H + L) (#711-545-152, 1:400) and Alexa Fluor 488 affinipure donkey anti-goat IgG (H + L) (#705-545-003, 1:400) were purchased from Jackson ImmunoResearch; Goat anti-mouse IgG (H + L) conjugated with HRP (#31430, 1:20000), goat anti-rabbit IgG (H + L) conjugated with HRP (#31,460, 1:20000) were purchased from Thermo Fisher Scientific.

Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, blocked with 1% BSA (bovine serum albumin) in PBS (blocking solution) for 30 min, and permeabilized with 0.1% Triton X100 for 3 min. Primary antibodies were diluted in blocking solution and incubated with cells at room temperature for 1 h. After three washes with PBS, cells were incubated for 1 h with secondary antibodies (diluted in PBS to final concentration 4 μg/ml). After three washes with PBS, cell nuclei were stained with Hoechst. The primary antibodies used for immunocytochemistry were rat anti-MBP (Serotec) used to measure OPC differentiation, and rabbit anti-AcH3K14 (against acetylated lysine 14 in histone H3, Millipore). Secondary antibodies were goat anti-rabbit IgG Alexa Fluor 488, and rabbit anti-rat IgG Alexa Fluor 488 (Invitrogen).

2D cultured cells were fixed with 4% (v/v) paraformaldehyde. 3D cultured cells were fixed as in 2D, but CaCl₂ was added to the solution to keep hydrogel disc integrity. Cells were further permeabilized and blocked in phosphate buffered saline (PBS), or instead in tris buffered saline with calcium chloride (TBS-CaCl₂) for 3D discs, containing 5% (v/v). Normal Goat Serum (NGS) (Biosource) and 0.2 % (v/v) Triton X-100 (Sigma). Primary antibodies were diluted in PBS or TBS-CaCl₂ containing 1% (v/v) NGS and 0.15% (v/v) Triton X-100, and incubated overnight in a humid chamber at 4°C. The following primary antibodies were used: rabbit anti-GFAP (1:500, Dako), mouse anti-vimentin (1:100, ThermoScientific), mouse anti-NG2 (1:100, Abcam), rat anti-MBP (1:500, AbD Serotec) rabbit anti-TAU (1:100, Sigma), mouse anti-CSPG (1:200, Millipore). Secondary antibodies Alexa-Fluor 488, 568, 594, and 660 were applied for 1 h at RT and subsequently treated for nuclear counterstaining at RT with Hoechst (Molecular Probes) at 2 μl.ml⁻¹. 3D samples were then observed under confocal microscope.

Cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 1% bovine serum albumin in PBS and 0.1% Triton-100 (blocking solution) for 1 h. Primary antibodies (rat anti-MBP, 1:200 dilution, Serotec) were diluted in blocking solution and incubated at room temperature for 1 h. Samples were washed 3 times with PBS and incubated with secondary antibodies (rabbit anti-rat IgG Alexa Fluor 488, 1:200 dilutions, Invitrogen) in PBS for 1 h, followed by washing and staining of nuclei with Hoechst 33342 at a 1:1000 dilution for 5 min.