小中大Double label ZO-1 immunofluorescence and apoptosis staining (tissue culture cells).
Rinse coverslips 1X in PBS.
Fix in -20oC methanol for 20 minutes
Keep Coplin jars in ethanol-dry ice bath.
Wash 3X 5 minutes in PBS.
Block with 10% goat serum in PBS for 30 minutes at RT.
Incubate with primary antibody.
Rabbit polyclonal anti-ZO-1 from ZYMED, 1:200 in PBS + 10% goat serum. Incubate for 1hr at 37oC in humid chamber.
Wash 3X 20 minutes in PBS.
Postfix in 4% paraformaldehyde for 10 minutes at RT.
Use 16% methanol free paraformaldehyde from Polysciences, diluted 1:4 in 1XPBS. Best done by using 4ml 10X PBS + 1 ampoule of 16% paraformaldehyde (10ml) and adjust the volume to 40ml by DI water.
Wash in PBS 1X5 min.
Block aldehydes in 100mM glycine in 0.5X PBS (pH 8.0) for 30 min. at RT.
Wash in PBS 2X5 min.
Block with 10% goat serum in PBS for 30 minutes at RT.
Incubate with secondary antibody.
Anti-rabbit IgG-Texas red conjugate (ICN), 1:100 in PBS + 10% goat serum. Incubate for 1hr at 37oC in humid chamber.
Incubate in 0.2% Triton X-100 in PBS for 5 min. at RT.
Wash 3X 20 minutes in PBS.
Remove excess liquid by tapping the slides and touching the edge of slides to absorbent paper.
Cover cells with 100 µl equilibration buffer and a piece of parafilm, to prevent drying and to spread the buffer evenly. Incubate at RT 5-10 min.
Remove parafilm and equilibration buffer from slides and touch the edge of slides to absorbent paper.
Incubate slides with labeling mix at 37oC for 60 min. covered with parafilm.
Labeling mix for a single reaction: 45 µl equilibration buffer, 5 µl nucleotide mix and 1 µl TdT enzyme. Multiply volumes with the number of samples.
Wash slides in 2XSSC for 15 min. at RT.
Immerse slides in Hoeshct solution for 10 min. at RT (1:5000 dilution of 1mg/ml stock in PBS).
Wash slides 3 X 5 min. in PBS.
Mount slides using anti-fade from Molecular probes.
If your procedure was successful, your result should look something like this
IEC-6 rat small intestinal epithelial cells were grown on glass coverslips and processed as described above. The three individual images in the following represent pseudo-colored fluorescence micrographs of nuclear stain with Hoeshct(top ), apoptosis stain with TUNEL staining (middle) and ZO-1 indirect immunofluorescence (bottom). The last image is a digital overlay of the three individual images. Staining was prepared by Luba Adler, MS.
