Smi 31

Hippocampal explants were fixed with 4% paraformaldehyde in PBS for 10 minutes and rinsed with PBS three times. Following permeabilization with 0.2% Triton X-100 in PBS, the cells were blocked with 10% fetal goat serum and 3% BSA for 30 minutes. 1 : 1000 dilution of SMI-31 (Abcam Inc., Cambridge, MA) and/or 1 : 500 dilution of MAP2 (Abcam Inc., Cambridge, MA) in BSA was applied for 1 hour at room temperature, followed by three washes in PBS. Fluorescently labeled secondary antibody (AlexaFluor-488 and AlexaFluor-594, Life Technologies) was then applied for 1 hr at 37°C, followed again by three washes in PBS.

Sections were stained for myelin with the use of luxol fast blue stain (LFB; Abcam) and for axons using immunohistochemical staining for phosphorylated neurofilaments (SMI-31; Abcam; Budde et al., 2007 (link)). Immunohistochemical staining for SMI-31 was performed as described previously (Budde et al., 2007 (link)). After accepting antigen retrieved with the use of citrate antigen retrieval solution (P0081; Beyotime), the tissue sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The sections were then incubated in 1:20 goat serum for 30 min, rinsed, and incubated overnight at 4°C with a 1:200 dilution of the primary antibody SMI-31. After three washes in PBS, the sections were incubated for 90 min with biotinylated goat anti-mice immunoglobulin G antibody (SP-9002; Zhongshan Golden Bridge Biotechnology). After another three PBS washes, the brain sections were incubated with avidin biotinylated horseradish peroxidase (SP-9002; Zhongshan Golden Bridge Biotechnology) for 90 min. The brain sections were rewashed three times in PBS and then incubated with diaminobenzidine and hydrogen peroxide (ZLI-9018; Zhongshan Golden Bridge Biotechnology). The nucleus was stained with hematoxylin. The sections were then rinsed in water for 10 min, dehydrated, and covered with a coverslip for microphotography.

Sections were labelled for 200 kDa phosphorylated neurofilament using SMI‐31 (Abcam; ab24570, 1:1000). Sections were quenched for 20 min in methanol 1% H₂O₂ to block endogenous peroxidase activity followed by citrate pretreatment as described above. Sections were blocked using 10% NHS and incubated with primary antibody overnight at 4°C before continuing with appropriate secondary antibody, ABC and DAB reaction as above.