Gb21302

Immunofluorescence staining was performed on NRCMs grown on coverslips. NRCMs were fixed with 4 % paraformaldehyde for 15 min and blocked with 3 % BSA in PBS for 30 min at room temperature. For α-actinin staining, NRCMs were incubated at 4 °C for overnight with a rabbit anti-α-actinin antibody (GB111556, Servicebio, Wuhan, China) and a corresponding anti-rabbit Cy3 antibody (GB21302, Servicebio, Wuhan, China) for 1 hour at room temperature. The image was obtained via the fluorescence microscope. The surface area of NRCMs were depicted using Image J. For co-localization of USP38 and TBK1, NRCMs were incubated with rabbit anti-USP38 at 4 °C for overnight, then incubated with corresponding anti-rabbit HRP-labled antibody for 1 hour, followed by Cy3-Tyramide (GB1223, Servicebio, Wuhan, China) for 1 hour at room temperature. Subsequently, NRCMs were incubated with rabbit anti-TBK1 antibody at 4 °C for overnight, then incubated with anti-rabbit Alexa Fluro 488 antibody (GB25303, Servicebio, Wuhan, China) for 1 hour at room temperature. The nucleus was stained with DAPI (GB1012, Servicebio, Wuhan, China) for 10 min. The image was obtained via the confocal scanning microscope (NIKON Eclipse TI, Tokyo, Japan).

Tissue paraffin sections were dewaxed and subjected to antigen retrieval treatment. After washing and blocking, the slides were incubated with the following primary antibodies: CD68 (1:200) (ab213363, Abcam, UK), fgl2 (1:200) (H00010875-M01, Abnova, Taiwan), and F4/80 (1:200) (ab6640, Abcam, UK). The subsequent secondary antibodies were Cy3-conjugated AffiniPure goat anti-rat IgG (1:200) (GB21302, Servicebio, China) and fluorescein isothiocyanate-labeled goat anti-mouse IgG (1:200) (GB22301, Servicebio, China). DAPI (G1012, Servicebio, China) was used for nuclear staining. The sections were observed under a fluorescence microscope (BX53, OLYMPUS, Japan). Three sections and 3-5 different positive microscopic fields in each section were chosen for semiquantitative analysis. Fluorescence intensity was analyzed by ImageJ software.