Ab227805

Cells were fixed in 4% paraformaldehyde for 15 min, followed by three washes of DPBS, blocked and permeabilized in PBS containing 0.1–0.2% Triton X-100 and 10% horse serum. The coverslips were incubated with primary antibodies; for NPCs: rabbit anti-PAX6 (CST, mAb#60433, 1:250) and mouse anti-NESTIN (CST, mAb#33475,1:2000); for Neurons: chicken anti-MAP2 (Abcam, ab92434, 1:500), rabbit anti-TBR1 (Abcam, ab183032, 1:250), rabbit anti-VGLuT1 (Abcam, ab227805, 1:500) and Mouse anti-GABA (Abcam, ab86186, 1:400) in the blocking solution overnight at 4 °C. On the next day, they were washed in DPBS and incubated with DAPI (Abcam, ab228549, 1:1000) and corresponding secondary antibodies (Abcam, ab150084, ab150117, ab175711, 1:250) for 60 min at room temperature. Then the coverslips were washed three times, mounted on glass slides using Fluromount-G (mounting medium), and dried overnight while being protected from light. Fluorescence signals were detected using a Leica THUNDER imager and analyzed using ImageJ and MATLAB.

The mouse hippocampus was lysed by radioimmunoprecipitation lysis buffer (Solarbio, Beijing, China, R0010) with a protease phosphatase inhibitor mixture (Solarbio, P1260). A BCA kit (Solarbio, PC0020) was applied to quantify the total protein concentration. Protein samples (40 μg per lane) were separated by 10% SDS-PAGE (Solarbio, P1200) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA, IPFL00010). The membranes were blocked with 5% skim milk (Solarbio, D8340) at room temperature (1 h) and subsequently immunoblotted with primary antibodies overnight at 4 °C: IL-33 (R&D, MND, USA, 1:500, AF3626), glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, UK, 1:1000, ab7260), vesicular glutamate transporter 1 (vGlut1) (Abcam, 1:1000, ab227805), postsynaptic density protein-95 (PSD95) (Abcam, 1:1000, ab13552) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, 1:2000, ab8245). Next, 1X tris-buffered saline Tween (TBST) (Solarbio, T1081) was used to wash the membranes 3 times. They were then incubated with a secondary antibody at room temperature (40 min). Immunoreactivity was detected using enhanced chemiluminescence (Solarbio, P0018), and bands were measured using Image J analysis software (Version 1.50i, Rockville, MD, USA).

On day 9, at 12 hours after administering NTG/VEH, the mice were deeply anesthetized with 1% pentobarbital sodium, and the brain TNC tissues of the medulla oblongata were quickly harvested on ice, frozen in liquid nitrogen, and stored at − 80 °C. The TNC tissues were sonicated in RIPA lysis buffer, added with 1 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF, 20 mM NaF, and 2 mM protease inhibitor cocktail, and quantified using the Bradford method. Proteins were separated using 8%–12% SDS-PAGE gels and were transferred to the PVDF membrane (Millipore). The PVDF membranes were blocked with 5% milk for 2 hours at room temperature, and were incubated overnight at 4 °C with the following primary antibodies: anti-c-Fos (1:1000; ab222699 Abcam), anti-CGRP (1:200; sc-57,053 Santa Cruz), anti-VGluT1 antibody (1:1000; ab227805 Abcam) and anti-GAPDH (1:5000; YM3445 ImmunoWay). Then, the membranes were washed in TBST and incubated with goat anti-rabbit or anti-mouse secondary antibodies (1:1000; Boster) for 1 hour at room temperature. The bands were visualized with Chemiluminescent HRP substrate (Millipore) on Azure Biosystem C500 and quantified using ImageJ.

At 3 days, 2 weeks, and 2 months after sub-FS stimuli, rats were anesthetized and decapitated. Brains were quickly removed to separate the hippocampus and cortex on ice. After measuring protein content using a BCA Protein Assay Kit (P0012; Beyotime), proteins were separated using 10% and 12% SDS-PAGE gel (P0012AC; Beyotime) and transferred to a polyvinylidene fluoride membrane (220 mA, 60 or 100 min, respectively). Subsequently, these membranes were blocked with 5% skim milk for 3 h. After incubation with the primary antibodies anti-synapsin I (1:1 000; ab254349; Abcam), anti-PSD95 (1:1 000; ab2723; Abcam), anti-VGLUT1 (1:1 000; ab227805; Abcam), anti-TGF-β1 (1:1 000; ab179695; Abcam), anti-TSP-1 (1:1 000; A00667-1; Boster), anti-caspase-3 (1:1 000; 9662; Cell Signaling Technology), anti-cleaved caspase-3 (1:1 000; ab2302; Abcam), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1 000; AB-P-R-001; Goodhere) overnight at 4°C, and the horseradish peroxidase-conjugated secondary antibody (1:3 000; ZB-2301 and ZB-2305; Beijing Zhongshan) for 2 h at 37°C, the membranes were exposed (ImageQuant LAS 500, USA). Gel bands were analyzed using ImageJ (version 1.49; National Institutes of Health; United States) software and are presented as ratios to GAPDH.

The mice were transcardially perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde under isoflurane deeply anesthetized. The brain was collected in 4% formaldehyde for 24 h at 4 °C, followed by 30% sucrose dehydration for 48 h at 4 °C. Coronal 30 μm-thick sections were cut on a cryostat (CM1900; Leica, Wiesbaden, Germany). The sections were washed 3 times with PBS and blocked with 5% goat serum at room temperature for 1 h. Then, the sections were incubated with primary antibodies overnight at 4 °C: IL-33 (R&D, 1:200, AF3626), GFAP (Abcam, 1:500, ab7260), vGlut1 (Abcam, 1:100, ab227805), PSD95 (Abcam, 1:100, ab13552) and ionized calcium-binding adapter molecule 1 (Iba1) (Wako, 1:500, 019-19741). The sections were incubated with secondary antibodies (Alexa Fluor 488 and 594) for 1 h at 37 °C after washing with PBS 3 times, followed by DAPI staining for 8 min at room temperature. Then, the sections were washed with PBS 3 times and mounted with 70% glycerol.