The confocal microscope used below was a BIO-RAD MRC 1024 with a three line Kr/Ag laser, excitation lines: 488, 568, 647 nm. The acquisition software used was BIO-RAD LaserSharp ver 3.2. Analysis of files obtained was performed using LaserSharp processing software. Images were subsequently manipulated with Adobe Photoshop™
All tissues were freshly dissected and covered with OCT (Tissue Tek™, Miles Inc. U.S.A.) mounting fluid then placed in a foil or plastic tray; this was then floated on liquid nitrogen or liquid nitrogen/isopentane slurry to quick-freeze. The resultant block was sectioned in a Cryostat; 4-6µm sections were collected onto cleaned glass slides and air-dried briefly before immersing in acetone, 30 sec, -20°C. The slides were then transfered to moist chamber staining trays and labelled for two or three colour fluororescence
Two colour protocol
MTS antibodies were made up containing rabbit anti-cytokeratin antibodies at 1 in 20 and incubated at RT 20 minutes. Three 5 minute washes of PBS (phosphate-buffered saline) were then followed by the final 20 minute incubation with Alexa-488 conjugated anti-rat Ig at 1 in 400 and Alexa-568 conjugated anti rabbit Ig up at 1 in 600. After three 5 minute washes of PBS a drop of mounting fluid (DAKO™ Fluorescent mounting medium) was placed on each section and coverslipped.
Three colour protocol
Incubation times and treatments were the same as above but method differs in antibody addition and a blocking step inclusion was necessary after primary incubation. Briefly, the primary antibody rat ant- mouse was added, incubated, washed off and Alexa-488 conjugated anti-rat Ig followed in the same manner as above. Normal rat serum (diluted 1 in 10) was then added and left for 10 minutes. The next step was 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 was added. The slides were washed finally and mounted as before.