Anti neun

Brains were fixed in 4% paraformaldehyde, following by incubation in 30% sucrose for at least 24 h. Tissue sections (15 micrometers thick) were incubated for 1 h in a blocking solution consisting of 0.3% Triton X-100, and 5% goat serum. This solution was used for the dilution of both primary (anti-NeuN 1:400, Cell Signaling Technologies, Danvers, MA) and secondary antibodies. Sections were incubated in the primary antibody overnight (16 h). Slides were then washed three times (5 min each) with PBS and visualized with Cy3-labeled secondary antibody and coverslipped. At least seven sections were analyzed for each mouse.

Immunofluorescence staining was performed as previously described [44 (link)]. In brief, 15 μm-thick coronal sections were rinsed in PBS 4 times for 5 min. The sections were blocked for 60 min in blocking buffer solution (1×PBS / 5% normal serum / 0.3% Triton ^TM X-100). Primary antibodies used were against Anti-NeuN (rabbit monoclonal, diluted 1:500) and tyrosine hydroxylase (TH) (rabbit polyclonal, diluted 1:200) (both Cell Signaling Technology, USA). Incubation with primary antibodies were performed overnight at 4 °C in primary antibody dilution buffer (1×PBST with 5% BSA). After being washed with PBST, the sections were stained with the secondary antibody, Alexa Fluor^®-488 goat anti-rabbit (Invitrogen) (1:500). The sections were incubated for 1 h in the dark at room temperature, then stained with DAPI (Beyotime Institute of Biotechnoloy, Haimen, China) for 1 min to reveal the nuclei. The brain sections were examined under a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Brain samples of mice were fixed in 4% paraformaldehyde, sequentially dehydrated in 20, 25, and 30% sucrose solution, frozen in OCT compound, and then prepared as 15-μm slices. Slices were incubated with indicated primary antibodies overnight at 4 °C, followed by incubation with appropriate secondary antibodies conjugated with fluorescence and DAPI for 60 min at room temperature. The fluorescence microscope images were acquired by an A1R (Nikon) confocal microscope. Antibodies used were: anti-NeuN (Cell Signaling Technology, 94403 S), anti-GFAP (Proteintech, 16825-1-AP), anti-Iba1 (Wako, 019–19741), and Alexa fluor 594-conjugated goat anti-rabbit IgG (H C L) secondary antibody (Thermo Fisher Scientific, A-11012).

The antibodies used in this study were as follows: anti-MAP2 (Abcam, Cambridge, UK, ab32454, 1:1000); anti-ACAT1 (Cell Signaling Technology, Boston, MA, USA, #44276, 1:1000); anti-NeuN (Cell Signaling Technology, Boston, MA, USA, #24307, 1:1000); anti-Ki67 (Cell Signaling Technology, Boston, MA, USA, #9449, 1:500); anti-TPK1 (proteintech, Chicago, IL, USA, 10942-1-AP, 1:1000); anti-β-actin (ZSGB-BIO, Beijing, China, TA346894). CHA was obtained from Jiujiang Biochemical Engineering Technology Development Co. (Chengdu, Sichuan, China). CHA for cell assays was dissolved in dimethyl sulfoxide (DMSO) at an appropriate concentration in cell experiments, and CHA for injection was dissolved in normal saline at the desired concentration in animal experiments.
The short hairpin RNA (shRNA) sequence against ACAT1 was purchased from IgeBio (Guangzhou, Guangdong, China). The target sequence of ACAT1-#1-specific shRNA was 5′-CGAAATGAACAGGACGCTTATCTCGAGATAAGCGTCCTGTTCATTTCG-3′ and that of ACAT1-#2-specific shRNA was 5′-GCCTTTAGTCTGGTTGTACTACTCGAGTAGTACAACCAGACTAAAGGC-3′; control (PKO.1-puro) shRNAs were synthesized by IgeBio. siRNAs targeting TPK1 were purchased from KeyGEN BioTech (Nanjing, China). All plasmids were transfected using the Lipofectamine 3000 transfection reagent, following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).

The brain tissues were collected in each group following perfusion with ice‐cold PBS 1 day after MCAO, and fixed with 4% paraformaldehyde for 48 h, then cut into slices. The slices were incubated in 0.3% Triton X for 10 min and blocked with 5% fetal bovine serum for 60 min at room temperature. The slices were incubated with the following primary antibodies overnight at 4°C after rinsing with PBS thrice, anti‐VAPB (66191, Proteintech), anti‐PTPIP51 (20641, Proteintech), and anti‐NeuN (94403, Cell Signaling Technology). The slices were washed thrice and then incubated with an Alexa 488‐conjuated antibody (ANT024S; Millipore, Billerica, MA) or Alexa 594‐conjugated antibody (ANT029S, Millipore, Billerica, MA) for 2 h at room temperature. DAPI was used to stain cell nuclei after washing with PBS. We randomly selected three brain sections from the ischemic region of each animal and observed VAPB, PTPIP51, NeuN immuno‐positive cells in the penumbra with an automated fluorescence microscope (Olympus, Japan). The number of positively stained cells was calculated and averaged with ImageJ. Statistical analysis was performed using five mice in each group.