Cy3 labeled goat anti mouse igg

Immunofluorescence (IF) staining was constructed by the method described in many studies. The tissue was embedded in paraffin and cut into 5-µm sections beforehand. All slides were deparaffinized, rehydrated in xylene, 100%, 95%, 85%, 75% ethanol, and PBS, and constantly stained with 10% antigen retrieval solution for 10 min. BSA (1%, Sangon) as the blocking buffer was incubated at room temperature for 15 min. The primary antibody was added and incubated at 4°C overnight. Then, the secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin) were added to this procedure. The antibodies used in this section involved as follows: NF-κB p-p65 antibody (1:200, Affinity, Jiangsu, China), CD68 (1:50, Santa Cruz, Santa Cruz, CA, USA), iNOS (1:100, ABclonal), Cy3 labeled goat anti-mouse IgG (1:200, Invitrogen, Carlsbad, CA, USA), Cy3 labeled goat anti-rabbit IgG (1:200, Invitrogen), FITC labeled goat anti-mouse IgG (1:200, Abcam, Cambridge, UK). The immunofluorescence images were taken and preserved using an OLYMPUS-BX53 microscope (OLYMPUS, Tokyo, Japan).

The primary neurons were treated by 200 µl 0.1% Triton X-100 (Beyotime) with 0.1% sodium citrate at room temperature for 15 min. The TUNEL reaction solution was prepared by
the enzyme solution and label solution in the In Situ Cell Death Detection Kit (Red) (Roche, Basel, Switzerland) according to 1:9, then added into the cell plate and incubated at 37°C for 60
min in the dark environment. Cells were photographed under a microscope after the nuclei was stained with DAPI (Aladdin).
TUNEL combined with IF was used to detect neuronal apoptosis. Neurons are located by the primary antibody NeuN (dilution rate 1:200, Abcam) and secondary antibody CY3 labeled goat
anti-mouse IgG (dilution rate 1:200, Invitrogen). In Situ Cell Death Detection Kit (Green) (Roche) was used to detect apoptosis.

Brain tissue was embedded in paraffin and then sectioned. BV2 microglial cells were washed with phosphate buffered saline (PBS) and fixed with 4% buffered paraformaldehyde (Sinopharm). Brain tissue and cells were then incubated overnight at 4°C with primary antibodies: FERMT1 (1:100 dilution; 22215-1-AP; proteintech, Hubei, China), ionized calcium binding adapter molecule 1 (Iba1; 1:50 dilution; Sc-32725; Santa, Shanghai, China) and NF-κB p65 (1:100 dilution; A19653; ABclonal, Hubei, China). Secondary antibodies were added and incubated for 1 h at room temperature. Secondary antibody: FITC-labeled goat anti-rabbit IgG (1:200 dilution; ab6717; Abcam, Cambridge, MA, USA), Cy3-labeled goat anti-mouse IgG (1:200 dilution; A-21424; Invitrogen) and Cy3-labeled goat anti-rabbit IgG (1:200 dilution; A27039; Invitrogen). Finally, nuclei were stained with DAPI (D106471-5mg, Aladdin). Tissue and cells were observed under an inverted fluorescence microscope (Olympus).