Immunostaining of Mouse Craniofacial Tissues

Take cryosections of fixed mouse embryonic craniofacial tissues comprising neural crest cells.

Wash the sections in buffer containing a non-ionic detergent to remove the embedding medium and permeabilize membranes, allowing access to intracellular targets.

Add blocking solution to saturate non-specific binding sites.

Remove the excess blocking solution. Then, incubate with primary antibodies targeting nuclear antigens, such as transcription factors and proliferation markers.

Wash with buffer to remove unbound antibodies.

Incubate with fluorophore-conjugated secondary antibodies to bind to the antigen-bound primary antibodies.

Remove the unbound antibodies using buffer.

Add an anti-fade medium with a fluorescent nuclear dye and cover with a coverslip.

The dye stains the nuclei, and the anti-fade medium protects fluorescent signals during imaging.

Image the sections under a fluorescence microscope to assess nuclear marker expression and localization, indicating neural crest cell proliferation and signaling activity.