Lm3050s

Pups were anesthetized and perfused with 0.1 M PBS followed by 4% formaldehyde solution (PFA) at 72 h post HI. The brains were removed and post-fixed (4% PFA, 4°C, 24 h), then transferred into a 30% sucrose solution. The brains were then sectioned at 10 mm thickness with a cryostat (Leica LM3050S) for Fluoro-Jade C^{30 (link)}, immunofluorescence staining, and 30 mm for Nissl’s staining.

Rats were anesthetized and perfused with cold PBS followed by 4% formalin at 24 h post HI. The brains were taken out and post-fixed overnight, then immersed in 30% sucrose until dehydrated and frozen in OCT. Coronal sections were cut at 10 μm thickness using a cryostat (Leica LM3050S). For nissl staining the slides were dehydrated in 95% and 70% ethanol for 1 min respectively, stained with 0.5% cresyl violet (Sigma-Aldrich, USA) for 2 min, and then dehydrated in 100% ethanol and xylene for 1.5 min consecutively. The percentage of brain tissue loss was calculated with the same equation for infarct area.
Immunofluorescent staining was performed as described previously (Ye et al., 2018 (link)). Briefly, after being permeabilized with 0.3% Triton X-100 and blocked by 5% donkey serum, sections were incubated with primary antibodies (FPR2, 1:100, Abcam, USA; Iba1, 1:100, Abcam, USA; GFAP, 1:100, Abcam, USA; IL-1β, 1:200, Abcam, USA; MPO, 1:200, Abcam, USA) at 4°C overnight. The next day sections were incubated with appropriate fluorescent dye-conjugated secondary antibodies (1:200) for 2 h at room temperature and mounted using Vectashield Antifade with DAPI (Vector Laboratories Inc., USA). The stained sections were captured under a fluorescence microscope Leica DMi8 (Leica Microsystems, Germany) and analyzed with Image Pro Plus software (Olympus, Melville, NY).

As previously described (Chen et al., 2011b (link)), pups were anesthetized and transcardially perfused with 0.1M PBS followed by 4% formaldehyde solution at 24h post HI. The brains were removed and postfixed (4% formaldehyde solution, 4°C, 24h), then transferred into a 30% sucrose solution for 2d. The brains were then sectioned at 10μm thickness with a cryostat (Leica LM3050S).
Slides were washed with 0.1M PBS 3 times and incubated in 0.3% Triton X-100 in 0.1M PBS for 30min at room temperature. They were then washed with o.1M PBS for 5min, 3 times and incubated with primary antibodies: sestrin2 (1:50, Proteintech Group, USA), vWF (1:1000, Abcam, USA), GFAP (1:1000, Abcam, USA), HIF1α (1:200, Santa Cruz Biotechnology, USA), MPO (1:100, Santa Cruz Biotechnology, USA) respectively (4°C, overnight). After washing with 0.1 M PBS (5min, 3 times), the slides were then incubated with secondary antibodies (Santa Cruz Biotechnology). Finally, slides were covered with DAPI (Vector Laboratories, Inc). Fluorescent microscope and Magna Fire SP system (Olympus) were used to analyze microphotographs.