Fitc goat anti rabbit igg

Brain slides were deparaffinized and placed in 0.01 M citrate buffer (pH 6.0). An autoclave was used to heat the citric acid buffer and keep it boiling for 2 min. Then, slides were endogenous peroxidase blocked with 3% H₂O₂ and incubated in 5% albumin bovine V (Solarbio) for 1 h. Subsequently, sections were incubated with the primary antibodies at 4 °C overnight, including IBA1 antibody (1:800, Proteintech, Wuhan, China), p-DRP1 (1:200, CST, USA), ASC (1:200, CST), and cGAS (1:200, CST). The next day, after washing with phosphate-buffered saline (PBS) buffer, the Tyramide signal amplification kit (AiFang biological, Hunan, China) was used for fluorescent double-label staining. Nuclei were counterstained with DAPI (Invitrogen, USA) for 1 min. Images were taken with a Pannoramic Scan (3Dhistech, Hungary, Budapest).
The cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The cells were permeated with 0.1% Triton X-100 and blocked in 1% BSA for 30 min before being incubated with an anti-rabbit TOM20 antibody (1:200, Proteintech) at 4 °C overnight. After washing with PBS, the sections were placed at room temperature with FITC goat anti-rabbit IgG (ABclonal, China) for 1 h and counterstained with DAPI for 1 min. Images were acquired with a Laser Scanning Confocal Microscope (Leica SP8, Germany).

Macrophages were stimulated with Mab1187 (10 μg/mL) for 24 h and then washed with PBS twice for 5 min, fixed with 4% paraformaldehyde for 15 min at room temperature in order to image mitochondrial morphology. The cells were incubated with 0.1% Triton X-100, blocked in 1% BSA for 30 min before being stained with an anti-rabbit TOM20 antibody (1:200, Proteintech, Wuhan, China) or a CoraLite®488-conjugated MLKL monoclonal antibody (1:250, Proteintech) at 4 °C overnight. After washing 3 times with PBS, the cells were incubated with FITC goat anti-rabbit IgG (ABclonal, China) for 1 h at room temperature. The nuclei were counterstained with DAPI for 1 min (Solarbio, China). Images were acquired with a Laser Scanning Confocal Microscope (Leica SP8, Germany).
Lung slides were placed in 0.01 M citrate buffer (pH 6.0). Heat citrate buffers until it boils and keeps it boiling for 10 min. Then the slides were washed with PBS thrice for 5 min and then blocked with 5% BSA for 1 h. Subsequently, sections were incubated with primary antibodies, including a CoraLite®488-conjugated MLKL monoclonal antibody (1:500, Proteintech) and anti-F4-80 (1:200). Next, similar fluorescence experiments were used for the lung sections. Images were taken with a Pannoramic Scan (3Dhistech, Hungary, Budapest).