IF staining was performed on 16-μm-thick frozen brain sections as described previously [53 (link)]. Briefly, the brain sections were permeabilized with 0.35% Triton X-100 for 15 min, blocked for 60 min at 37 °C with goat serum, then incubated with the matching primary antibodies at 4 °C overnight. The primary antibodies included mouse anti-caspase 1 (1:40, Santa Cruz, sc-56036), mouse anti-NeuN (1:200, MilliporeSigma, MAB377), mouse anti-GSDMDC1 (1:50, Santa Cruz, sc-393656), rabbit anti-NeuN (1:100, Proteintech, 26975-1-AP), and rabbit anti-α7nAChR (1:50, Bioss, bs-1049R). On the following day, the slides were incubated with corresponding secondary antibodies and 4′,6-diamidino-2-phenylindole. Subsequently, the slides were imaged using a microscope (Nikon, Tokyo, Japan) and analyzed using the ImageJ software.
TUNEL staining (Boster, Wuhan, China) was used to count the number of dead cells, following the manufacturer’s instructions as previously described [54 (link)]. The number of TUNEL-positive cells was statistically analyzed by a blinded observer using the ImageJ software.
TUNEL staining (Boster, Wuhan, China) was used to count the number of dead cells, following the manufacturer’s instructions as previously described [54 (link)]. The number of TUNEL-positive cells was statistically analyzed by a blinded observer using the ImageJ software.
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