Immunohistological Analysis of Mouse Thymus

Immunohistology was carried out as previously described [81 (link)]. In Brief, after euthanizing mice, the thymus was excised under aseptic conditions and frozen in optimal cutting temperature media (Tissue-Tek). Five-micron frozen sections were cut using a Microm HM 550 Cryostat (Thermo Scientific), collected on coated slides, fixed in 4% paraformaldehyde, washed with PBS, and blocked with appropriate sera in PBS. After incubating with appropriate antibodies, sections were washed and incubated with fluorescence dye-conjugated second antibodies and 1 μg/ml of DAPI (Sigma). Stained sections were washed and mounted under a coverslip using Fluoro-gel with Tris Buffer (Electron Microscopy Sciences, Hatfield, Pennsylvania) and examined using an Axio Observer fluorescence microscope (Zeiss).