Alexa fluor 488 goat anti mouse

The sections of clinical specimens were incubated with the primary antibody against p-MLKL (Abcam) and CD68 (Abcam), while murine sections were incubated with antibodies for p-MLKL (Invitrogen) and F4/80 (Abcam) at 4°C overnight. After rinsing with phosphate-buffered saline (PBS), the sections were labeled with fluorescently labeled secondary antibodies, including Alexa Fluor 594 goat anti-rabbit (ZSGB-BIO), Alexa Fluor 488 goat anti-mouse (ZSGB-BIO), and Alexa Fluor 488 goat anti-rat (Abcam). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich). Fluorescence images were captured using a fluorescence microscope (Zeiss).

The mice were anaesthetised with 2% isoflurane and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde until their limbs stiffened. The brains were fixed in 4% paraformaldehyde for at least 24 h at 4 °C and then immersed in 30% sucrose solution for another 24 h. A freezing microtome (CM1850, Leica, Hesse-Darmstadt, Germany) was used to prepare 20-µm-thick brain slices.
Then, brain slices were labelled with primary antibodies at 4 °C overnight, including C-Fos (1:500, mouse, GTX16902, Neobioscience, Shenzhen, China), NALCN (1:500, rabbit, ASC-022, Alomone, Jerusalem, Israel), and C-Fos (1:500, rabbit, ab222699, Abcam, Cambridge, UK). After overnight incubation, the primary antibody was washed with PBS and incubated with the secondary antibody, including Alexa Fluor 488 goat anti-mouse (1:500, ZF-0512, ZSGB-BIO, Beijing, China), Alexa Fluor 555 donkey anti-mouse (1:500, A-31570, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Alexa Fluor 550 goat anti-rabbit (1:500, 550045, ZEN-BioScience, Chengdu, China) for 2 h. All images were taken with a Zeiss Axio Imager Z.2. and analysed using ImageJ software V1.8.0 (National Institutes of Health, Bethesda, MA, USA).