Immunohistochemical

ImmunohistochemistryMice 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 quantificationContralesional 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 o