Ab150108

The spinal cords were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into slices of 4 μm thickness. After deparaffinization and rehydration, hematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining were performed as previously described [19 (link)]. For fluorescence immunohistochemistry, the sections were incubated overnight with mouse monoclonal primary antibody against IBA1 (Servicebio, GB12105, 1:150) at 4 °C, followed by staining with a donkey anti-mouse IgG secondary antibody (Abcam, ab150108, 1:200). For fluorescence immunocytochemistry, the cells were harvested after gently repeated blowing, centrifuged at 1000 rpm, fixed with 4% PFA, permeabilized with 0.1% Triton ×100, and incubated with primary mouse monoclonal anti-MGO antibody (Abcam, ab243074, 1:200) at 4 °C overnight, followed by staining with donkey anti-mouse IgG secondary antibody (Abcam, ab150108, 1:200). All images were captured using Olympus BX51 or LEICA DMi8 microscope.

After deep anesthesia, the rats received 200 ml of 0.9% saline followed by 200 ml
of 4% paraformaldehyde. The lumbar enlargement (L4-L6) was harvested and
postfixed in 4% paraformaldehyde for 6–8 h, then transferred to a 30% sucrose
solution and stored at 4°C, dehydrated and allowed to sink to the bottom. Twenty
micrometer-thick frozen sections were cut with a freezing microtome and blocked
in 10% donkey serum and 0.4% Triton X-100 in 0.01 m PBS for 2 h. The
sections were divided into six groups and incubated with primary antibodies at
4°C for 24 h. After washing three times with PBS, the sections were incubated
with secondary antibodies for 2 h at room temperature. The primary antibodies
used in the present study were rabbit anti-p-PAK1 (1:1,000, Affinity, AF3424),
mouse anti-NeuN (1:1,000, MAB377, Millipore), goat anti-glial fibrillary acidic
protein (GFAP) (1:500, Abcam, ab53554), goat anti-Iba1 (1:300; Abcam, ab48004),
and rabbit anti-Rac1 (1:200, Affinity, AF4200), Donkey Anti-Rabbit IgG H&L
(Alexa Fluor® 488) (1:500, Abcam, ab150073), Donkey Anti-Goat IgG H&L (Alexa
Fluor® 594) (1:500, Abcam, ab150132), Donkey Anti-Mouse IgG H&L (Alexa
Fluor® 594) (1:500, Abcam, ab150108). Details can be found in a previous
work.^{36 (link)} Images were obtained under a confocal laser scanning
fluorescence microscope (Olympus FV1000, Japan).