For immunofluorescence studies, scales were separated from the portions of skin by prising them
out of scale pouches using fine tungsten needles. Then the procedure described by Byers et al.
(1980) was used with the following modifications. A solution of 3% formaldehyde in teleost
phosphate-buffered saline (TPBS) (Grimstone & Skaer, 1972) was used for fixation. Microtubules
were decorated with a monoclonal rat anti-yeast-tubulin (YOL 1/34) (Serotec Ltd, Bicester, UK),
which was used in conjunction with fluorescein isothiocyanate (FITC)-conjugated sheep anti-rat
immunoglobulin (Serotec Ltd) as secondary antibody. Scales were mounted in a mixture of 9 parts
glycerol: 1 part TPBS (v/v) (with diazobicyclo-octane at a final concentration of 2 mg ml-1 added
to reduce the rate of photobleaching) for examination with a Leitz Ortholux fluorescence
microscope using a X50 water immersion objective (n.a. 1-0). Kodak Tri-X film was used for
photography.