Ab45953

Macrophages were washed with PBS three times. Four percent fixative solution (Solarbio, P1110) was added to the Petri dish for 10 min. Then, the solution was removed and cells washed three times. Next, 0.5% Triton X-100 (Solarbio, T8200) was added to the dish for 10 min. The solution was removed and cells washed three times. Five percent BSA (Solarbio, A8010) was added to the dish, and it was incubated for 1 h at room temperature. Then, primary antibodies were added to the M0 (GLRX1: 1:500, Abcam, ab45953; CD11b: 1:100, Proteintech, 66519-1-lg) and M2 macrophages (GLRX1: 1:500, Abcam, ab45953; CD163: 1:100, Abcam, ab156769) (18 (link)), respectively, and they were incubated overnight at 4°C. The solution was removed and cells washed three times. Secondary antibodies (DyLight 488 goat antirabbit polyclonal antibody, Abcam, ab96899, 1:200; DyLight 594 goat antimouse polyclonal antibody, Abcam, ab96881, 1:200) were used for 1 h at room temperature. The solution was removed and cells washed three times. Prolong™ Diamond Antifade Mountant with DAPI (Invitrogen, P36962) was added to the dish, and photos were taken with confocal microscopy.

Paraffin-embedded samples were obtained from the CGGA sample bank. First, 5-µm sections were cut for IHC staining. Samples were deparaffinized in an oven at 65°C for 1 h. Then the samples were rehydrated in decreasing concentrations of alcohol. IHC analysis with GLRX1 (Abcam, ab45953,1:1000) and CD11b antibodies (Proteintech, 66519-1-lg, 1:1000) was conducted according to standard procedures. Photos were taken with an optical microscope.