Ab184247

The trigeminal ganglia from mice were first homogenized by a tissue homogenizer, and then samples were lysed by RIPA (Beyotime, China) containing 1% PMSF (Solarbio, China). The cells were directly lysed by RIPA with 1%PMSF. The proteins were boiled at 100 °C for 10 minutes. The proteins were isolated by 10% SDS-PAGE gel (Shanghai Epizyme Biomedical Technology Co., Ltd., China) and transferred to polyvinylidene fluoride (PVDF) membrane (Solarbio, China). After blocking with 5% skim milk, PVDF membrane was incubated with primary antibody overnight at 4 °C. The primary antibodies used in this experiment mainly include Runx2 (Abcam, ab192256, 1:1 000), Sema3A (Abcam, ab23393, 1:1 000), NGF (Huabio, ET1606-29, 1:1 000), Rock2 (Huabio, ER1706-48, 1:500), Drp1 (Abcam, ab184247, 1:1 000), Mfn2 (Huabio, ER1802-23, 1:1 000), and GAPDH (Huabio, ET1601-4, 1:2 000). HRP-conjugated secondary antibody (1:2 500, Huabio) was used to combined primary antibody. Super ECL Plus kit (US Everbright, China) was used to visualize proteins, and images was obtained by a Chemi Doc Touch Imaging System (Bio-RAD, USA).

Tissue samples were decalcified in 10 mM ethylene diamine tetraacetic acid (EDTA) for 3 weeks, then dehydrated overnight with 15% and 30% sucrose, respectively, and finally embedded with optimal cutting temperature compound (OCT) (Sakura, Japan). Tissue sections of 10-μm thickness were prepared with a Leica cryostat. Cell samples were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.3% Triton X-100 for 10 minutes and blocking with 3% BSA for 1 hour. Tissue sections or cells were incubated overnight with primary antibody at 4 °C. The primary antibodies used in this experiment are as follows: CD31(Abcam, ab182981, 1:200), CGRP (Abcam, ab36001, 1:200), Runx2 (Abcam, ab192256, 1:200), Sema3A (Abcam, ab23393, 1:200), Rock2 (Huabio, ER1706-48, 1:200), Drp1 (Abcam, ab184247, 1:200), and Mfn2 (Huabio, ER1802-23, 1:200). After washing the primary antibody, samples was incubated in the secondary antibodies for 2 hours. The secondary antibodies used are as follows: goat anti-rabbit Alexa Fluor 555 (Huanio, HA1123, 1:200), goat anti-rabbit Alexa Fluor 647 (Huabio, HA1123, 1:200), donkey anti-goat Alexa Fluor 555 (Abcam, ab15011). Immunofluorescence images were collected by a Nikon confocal microscope (Nikkon, N-Strom & A1, Japan).