Bx50 bx fla dp70

The vivo immunofluorescence analysis was prepared as follows. Briefly, the brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm sections which were stained with primary antibodies (diluted 1:100) and appropriate secondary antibodies (1:500dilution). Normal rabbit IgG or normal mouse IgG was used as a negative control (data not shown). Finally, sections were observed by a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan), and the relative fluorescence intensity was analyzed by use of Image J program.
The in vitro multiple labeling immunofluorescence analysis was also performed. In brief, cells were fixed in 4 % paraformaldehyde for 15 min at room temperature and blocked in 5 % bovine serum albumin (Biosharp, Hefei, China) for 30 min, then incubated with primary antibody overnight at 4 °C. Then, cells were washed with PBST for three times and incubated with another antibody overnight at 4 °C. Repeated the above process until the total primary antibodies all used. Finally, cells were washed with PBST and incubated with corresponding appropriate secondary antibodies at 37 °C for 30-60 min. The fluorescence images were captured using a laser scanning confocal microscope (ZEISS LSM 880, Carl Zeiss AG, Germany).

The brain samples were fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 4 μm sections. The cultured neurons were fixed in 4% paraformaldehyde. Then, the sections and neurons were incubated with primary antibody against RIP1, RIP3, MLKL and caspase-8 and secondary antibodies. Normal rabbit IgG, normal mouse IgG and normal goat IgG were used as negative controls for the immunofluorescence assay (data not shown). Nuclei were stained with DAPI mounting medium. Finally, the sections and neurons were observed in a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan). The relative fluorescence intensity was analyzed with the Image J program. The quantitative analysis was performed by an observer who was blinded to the experimental group.

PI staining in vivo to identify microglial necroptosis was conducted as previously described (31 (link)). For all experiments, PI (Beyotime, Shanghai, China) was administered intraperitoneally (10 mg/kg) 1 h before the mice were sacrificed. The brain tissue was cryoprotected by immersion in 15 and 30% sucrose solution. Then, brain sections (10 μm) were cut along the anterior–posterior lesion and placed on poly-l-lysine-coated glass slides. Sections were visualized under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co.). PI+/Iba-1+ cells were counted by observers blinded to the experimental groups. Necroptotic microglia were counted in six microscopic fields per section, and the average number in each section was calculated.

pSIVA-IANBD was dialyzed into medium and applied to the neurons at 10–20 ul/ml concentration, according to the protocols. In this test, fluorescently tagged annexin binds to externalized phosphatidylserine exposed on cell membranes, allowing for monitoring changes that occur at different stages of apoptosis in living cells (33 (link)). After 15 min incubation, the cells were fixed and immunostained using MAP2 or TMEM16F antibody. Then, they were incubated with Alexa Fluor 555 conjugated anti-mouse antibody, as described above. In particular, the exposed PS binding with pSIVA did not need extra staining with secondary anti-body and could be observed directly using a green fluorescence filter set. Nuclei were stained with DAPI mounting medium. Cells were observed by a fluorescence microscope (OLYMPUS BX50/BXFLA/DP70; Olympus Co., Tokyo, Japan).

The mitochondrial membrane potential of primary neurons was detected using the mitochondrial membrane potential detection kit (JC-1; Beyotime Biotechnology Inc.). JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential [50 ]. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form J-aggregates, and generates red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix. Therefore, JC-1 remains monomeric and produces green fluorescence. Dye solution was diluted with dye buffer and added to the culture plate for staining at 37 °C for 20 min. Neurons were washed twice with dye buffer and then observed under fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus).

Neurons were fixed in 4% paraformaldehyde for 10 min, stained by Hoechst 33258 at room temperature for 15 min, and observed by a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan). Normal neuron nuclei were homogeneously stained blue, while the nuclei of apoptotic neurons displayed nuclear fragmentation or chromatin condensation. Apoptosis was assessed in ≥300 cells. The quantitative analysis was performed by an observer who was blind to the experimental group.

Based on the manufacturer's protocol for the in situ Cell Death Detection Kit (11684795910, Roche, Mannheim, Germany), TUNEL staining was performed to detect cell death in brain sections. Six random fields from each group were observed using a fluorescence microscope (Olympus BX50/BX-FLA/DP70; Olympus). Image J software was used to analyze the positive rate of TUNEL staining by an observer who was blind to the experimental groups.