Exploring HMGB1 and Microglia Activation in CUMS-Induced Rats

To explore the effects of PNGL on HMGB expression and IBA1-marked microglia activation in CUMS-induced rats [42 (link),43 (link)], immunofluorescence staining was performed as previously described [7 (link),8 (link),44 (link),45 (link)]. Briefly, after the micro slides were deparaffinized, dewatered, and restored with a citrate-EDTA antigen retrieval solution (P0086, Beyotime, Shanghai, China) for 20 min at 95 ℃, they were cooled down and washed with PBS three times (10 min per time), blocked with 5% goat serum albumin at room temperature for 60 min, and then incubated overnight with an anti-HMGB1 antibody (ab79823, 1:500 in dilution) and IBA1 (ab15690, 1:400 in dilution) at 4 °C. Subsequently, they were incubated with a TRITC-conjugated goat anti-rabbit IgG at a 1:100 dilution (CW0160, CWBIO, Beijing, China) and a Alexa Fluor 488-labeled goat anti-mouse IgG (P0188, Beyotime, Shanghai, China) for 2 h at room temperature, and then counterstained by DAPI (5 μg/mL) for 10 min. Images were observed using fluorescence microscopy (Leica, Germany Q9). The fluorescence intensity was evaluated by the ImageJ 1.44p software.