Flourescent Immunostaining for Confocal Microscopy



Labelling Method

Freshly dissect tissue and cover with OCT (Tissue Tek™, Miles Inc. U.S.A.) mounting fluid then placed in a foil or plastic tray

Then float on liquid nitrogen or liquid nitrogen/isopentane slurry to quick-freeze.

Section resultant block in a Cryostat

Collect 4-6µm sections onto cleaned glass slides and air-dry briefly before immersing in acetone, 30 sec, -20°C.

Then transfer the slides to moist chamber box staining trays and label for two or three colour fluorescence

Two colour protocol

Make up MTS antibodies containing rabbit anti-cytokeratin antibodies at 1 in 20 and incubate at RT for 20mins.

Three 5 minute washes of PBS (phosphate-buffered saline) followed by the final 20min incubation with Alexa-488 conjugated anti-rat Ig at 1:400 and Alexa-568 conjugated anti rabbit Ig up at 1:600.

After three 5 minute washes of PBS, place a drop of mounting fluid (DAKO™ Fluorescent mounting medium) on each section and a coverslip.

Three colour protocol

Incubation times and treatments are the same as above but method differs in antibody addition and a blocking step inclusion is necessary after primary incubation.

Briefly, the primary antibody rat anti-mouse is added, incubated, washed off and Alexa-488 conjugated anti-rat Ig following in the same manner as above.

Normal rat serum (diluted 1:10) is then added and left for 10mins.

The next step is a combined biotinylated rat anti-mouse antibody with rabbit anti-cytokeratin.

After washing as before either CY5 streptavidin and Alexa-568 conjugated anti rabbit Ig or Alexa-568 streptavidin and Cy5 anti-rabbit is added.

The slides are then washed finally and mounted as in the two colour protocol.