Immunofluorescence Staining of the Manchette and Developing Sperm Flagella in Mouse

Immunofluorescence staining is a technique that permits visualization of components of various cell preparations. During spermiogenesis, cargo proteins are transported through the intra-manchette transport (IMT) to the basal bodies; then sperm flagella are assembled by intraflagellar transport (IFT). Due to the big complexes formed by IMT and IFT components, it has been challenging to visualize these components in tissue sections. Testicular tissue is digested by a combination of enzymes (DNAse 1 and Collagenase IV) and fixed by paraformaldehyde and sucrose. After being permeabilized with Triton X-100, testicular cells are incubated with special primary antibodies to detect the specific components in the manchette and developing tails.