Picrosirius red (Abcam; Cat# ab245887) and immunofluorescence staining were performed by standard methods using 8 μm paraffin sections. Antibodies used for immunofluorescence staining were F4/80 (Abcam; Cat# ab6640; 1:100), Ly6g (Abcam; Cat# ab25377; 1:100), phospho-histone H3 Ser10 (Millipore; Cat# 06-570; 1:200), anti-Claspin (Thermofisher; Cat# PA5-102840; 1:100), anti-MCM2 (Abcam; Cat# ab4461; 1:100), anti-Orc2 (Thermofisher; (Cat# PA5-70227; 1:100), and anti-ATR (Proteintech; Cat# 19787-1-AP; 1:100).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).