Superfrostplus adhesion glass

For immunofluorescence, spleens were embedded and frozen in OCT, sectioned at 15 μm and mounted on SuperFrostPlus Adhesion glass (Thermo Fisher Scientific). Sections were dehydrated using silica beads, fixed with 4% paraformaldehyde for 10 min and washed with PBS. Samples were blocked using 5% normal goat serum for 2 h before staining. Samples were incubated with antibodies against B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience) and F4/80 (BM8, Biolegend) diluted in 5% NGS for 2 h at room temperature in the dark. After staining, samples were washed with PBS at least three times. Samples were then mounted using ProLong Gold Antifade Mountant (Invitrogen) and imaged using an inverted LSM780 microscope (Carl Zeiss) and a plan apochromat 63× NA 1.40 oil-immersion objective (Carl Zeiss). For haematoxylin and eosin (H&E) staining, organs were collected and fixed in 10% formalin. Fixed samples were embedded in paraffin and sectioned at 10 μm, mounted on SuperFrostPlus Adhesion glass and stained using H&E. Mounted samples were imaged using a Nikon SMZ1270 Stereo Microscope. Imaging data were analysed using Fiji (ImageJ) software (NIH).