Immunohistochemical

Immunohistochemistry
Mice were perfusion-fixed with 4% paraformaldehyde (PFA) at 3, 7, and 14 days post-injury. Brains were dissected and post-fixed in 4% PFA overnight at 4°C. Samples were immersed in 30% sucrose solution for 1−2 days at 4°C. Samples were subsequently embedded in Tissue-Tek OCT compound (Sakura Finetek) and stored at -80°C. Frozen brainstem samples were cut into 30 mm-thick coronal sections using a cryostat.
Sections were antigen-retrieved in 0.01 M citrate buffer (pH 7.0) and heated to 90°C in a thermostatic bath for 5 min. After antigen retrieval, sections were cooled for at least 30 min at room temperature. Sections were blocked with 10% normal goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 in PBS for 1.5 h at room temperature. Sections were then incubated with the following primary antibodies overnight at room temperature: guinea pig anti-VGluT1 (1:500; Synaptic Systems, 135304), rabbit anti-GluA1 (1:100; Abcam, ab183797), and rabbit anti-GluA2 (1:1000; Abcam, ab206293). The sections were then incubated with the following secondary antibodies for 2 h at room temperature: Alexa Fluor 488, 647 goat anti-rabbit, guinea pig IgG (1:500; Life Technologies, A11008, Invitrogen, A21450), and Alexa Fluor 594 streptavidin (1:400; Invitrogen, S11227). The antibodies were diluted in PBS containing 0.1% Triton X-100, 10% normal goat serum, and 1% bovine serum albumin. The sections were washed between tests with PBS and 0.05% Tween 20 in PBS. Finally, the sections were mounted with a fluorescence mounting medium (DAKO, S3023), and coverslips were added.

Histological quantification
Contralesional MdV Z-stack images (0.3 mm/step, 13−15 steps/image) were acquired from the stained sections using a confocal laser scanning microscope (Nikon, TiE-A1R). MdV was identified using the brain atlas by Paxinos and Franklin (Paxinos G and Franklin KBJ, 2001) and photographed at the BDA-positive varicosities. One to three images (XY size: 151.4 × 151.4 mm, resolution: 0.15 mm/pixel) were acquired per section, and six images per mouse were used for analysis.
The acquired images were threshold-processed for background elimination and quantitatively measured using ImageJ software (NIH). We identified triple-positive sites of BDA, VGluT1, GluA1, and GluA2. We then measured the puncta area of VGluT1 and GluA1 or GluA2 signals (at least four pixels). Additionally, we divided the areas of GluA1 and GluA2 by the VGluT1 areas at the same site to calculate the ratio of AMPA receptors to presynapses. All images were assigned file names with random numbers to exclude analyst bias.

免疫组织化学（Immunohistochemistry）
实验小鼠于损伤后3、7、14天采用4%多聚甲醛（paraformaldehyde, PFA）进行灌注固定。取脑组织解剖分离后，于4℃条件下用4% PFA后固定过夜。将样本置于4℃的30%蔗糖溶液中浸泡1~2天。随后将样本用Tissue-Tek OCT复合包埋剂（Sakura Finetek公司）包埋，并保存于-80℃冰箱。使用冰冻切片机（cryostat）将冰冻脑干样本切成30 mm厚的冠状切片。

切片采用0.01 M柠檬酸盐缓冲液（pH 7.0）进行抗原修复，并于恒温水浴箱中加热至90℃维持5分钟。抗原修复完成后，将切片在室温下冷却至少30分钟。随后用含10%正常山羊血清、1%牛血清白蛋白及0.3%曲拉通X-100（Triton X-100）的磷酸盐缓冲液（PBS）在室温下封闭1.5小时。接着将切片与以下一抗（primary antibodies）于室温下孵育过夜：豚鼠抗VGluT1（1:500；Synaptic Systems公司，货号135304）、兔抗GluA1（1:100；Abcam公司，货号ab183797）及兔抗GluA2（1:1000；Abcam公司，货号ab206293）。之后将切片与以下二抗（secondary antibodies）于室温下孵育2小时：Alexa Fluor 488、647标记的山羊抗兔IgG、豚鼠IgG（1:500；Life Technologies公司，货号A11008；Invitrogen公司，货号A21450），以及Alexa Fluor 594标记链霉亲和素（1:400；Invitrogen公司，货号S11227）。所有抗体均用含0.1% Triton X-100、10%正常山羊血清及1%牛血清白蛋白的PBS进行稀释。孵育过程中及孵育结束后，均使用PBS及含0.05% Tween 20的PBS对切片进行洗涤。最后，使用荧光封片剂（DAKO公司，货号S3023）对切片进行封片，并加盖盖玻片。

组织学定量（Histological quantification）
使用共聚焦激光扫描显微镜（confocal laser scanning microscope，尼康TiE-A1R型号）对染色后的切片采集对侧MdV的Z-stack图像：每步间距0.3 mm，每张图像包含13~15个扫描层。参照Paxinos与Franklin的脑图谱（Paxinos G and Franklin KBJ, 2001）定位MdV脑区，并对BDA阳性曲张体区域进行拍照。每个切片采集1~3张图像（XY尺寸：151.4 × 151.4 mm，分辨率：0.15 mm/像素），每只小鼠选取6张图像用于后续分析。

采集到的图像通过阈值处理消除背景噪声，并使用ImageJ软件（美国国立卫生研究院，NIH）进行定量分析。我们首先识别出BDA、VGluT1、GluA1及GluA2的三阳性共定位位点，随后测量VGluT1与GluA1或GluA2信号的斑点面积（至少包含4个像素）。此外，我们计算同一共定位位点处GluA1、GluA2的面积与VGluT1面积的比值，以得到AMPA受体（AMPA receptors）与突触前结构的相对比例。所有图像均采用随机编号进行命名，以排除分析者偏倚。