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.

Rinse the slides in 0.1% PBST three times for five minutes each, to wash out OCT and permeabilize the sections. Add 200 microliters of blocking solution to each slide and incubate at room temperature for 30 minutes. Then, remove the blocking solution without rinsing the slide. Add 100 microliters of primary antibody diluted in blocking solution to each slide, and incubate overnight at 4 degrees Celsius.

Rinse the slides with PBS three times for 10 minutes each at room temperature. Add 100 microliters of secondary antibody diluted in blocking solution, and incubate for one hour at room temperature, protected from light. Rinse in PBS three times for 10 minutes each at room temperature, still protected from light. To mount the slides, add two drops of anti-fade medium with DAPI on each slide. Cover with a coverslip and store at 4 degrees Celsius until ready to image.