Alexa 488

Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.5% Triton-X100 for 20 min then blocked with QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 1 h at room temperature. Cells were washed carefully with PBS for three times after each step. The fixed cells were incubated with anti-MUC1 antibody (CST, USA), anti-Flag (CST, USA) or anti-Lamin B1 (ProteinTech, Wuhan, China) for 2 h at room temperature. FITC-labeled rabbit anti-MUC1, anti-Flag or Cy3-labeled mouse anti-Lamin B1 were co-incubated with Alexa-488 or Alexa-555 secondary antibody (P0176 & P0190, Beyotime, Shanghai, China). Nuclei were stained with Hoechst 33342 (C1026, Beyotime, Shanghai, China). Finally, the cells were visualized and photographed under laser confocal microscopy (Nikon, Japan).

The protocol of immunofluorescence staining was performed according to Yu et al. (2014) (link). Briefly, C18-4 cells and Shp2 knockdown cells, which were cultured in a 48-well plate after treatment with CGA, were fixed with 4% formaldehyde for 15 min and washed with PBS three times for 3 min each. The cells were permeabilized with by 0.1% Triton X-100 (Solarbio, Beijing, China) for 15 min and blocked for 30 min with 1% BSA at 37°C. Then, the cells were incubated with primary antibodies specific against PLZF (1:200, Santa Cruz Biotechnology, California), SHP2 (1:100, Abways, Shanghai, China) and C-KIT (1:300, Biolegend, San Diego) for 12 h at 4°C. Alexa-488 (1:500, Beyotime Biotechnology, Shanghai, China) secondary antibodies were used to incubate cells for 1 h at 37°C. The negative control was stained with conjugated secondary antibodies alone: goat anti-rabbit IgG and goat anti-mouse IgG. The nuclei of cells were stained with DAPI (Beyotime Biotechnology, Shanghai, China) (Niu et al., 2016 (link)).

Immunofluorescence was used to detect microglial markers (Ibal-1), IL-1β, TNF-α, and their receptors and c-Fos. Tissue sections were made following routine perfusion sampling, dehydration, and fixation with paraffin-embedding. Coronal sections of Iba-1-labeled microglia are 8 µm thick; other labels are 5 µm thick. A cryostat (CM3050S; Leica Inc., Wetzlar, Germany) was used to obtain the TNC and then treated with Cy3 goat anti-rabbit secondary antibodies for 30 min at 25 °C, followed by incubation with relevant antibodies for 1 h at 37 °C. Double labeling was performed using two primary antibodies obtained from different genera. After washing, the slices were incubated at 37 °C for 0.5 h with a solution containing species-specific secondary antibodies conjugated to Alexa 488 (Beyotime, Shanghai, China). After washes, all sections were counterstained with DAPI.
The slides were scanned using Pannoramic MIDI (3DHISTECH; Budapest, Hungary) and measured using CaseViewer 2.3 software (3DHISTECH; Budapest, Hungary) at 40× magnification. The cell number and fluorescence intensity were observed by a morphological image analysis instrument (JD801, JSJD Tech Inc., Nanjing, China) as well as Image J (https://imagej.net/, accessed on 6 December 2020) in squares. The image analysis was performed by a blinded observer.