Immunofluorescence staining of cryostat sections

Wolf D. Kuhlmann

Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany

Laboratory Diagnostics & Cell Science, 56112 Lahnstein, Germany

The procedure of immunofluorescence localization of collagen types and laminin in frozen cut sections from oral mucosa is described. Tissue specimens may be obtained by surgery or from biopsy; fixation is optinonal, f.e. by aldehydes. Specimens are placed on a piece of aluminium foil (or in a special cryomold), covered by a drop of water soluble embedding medium such as Tissue-Tek®) and immediately frozen in liquid nitrogen cooled isopentane; frozen tissue blocks are tranferred to the cryo-chamber for sectioning. Frozen cut sections of 4-8 µm thickness are prepared with a cryo-microtome at -25°C to -30°C and using an anti-roll plate. For improved adherence of tissue sections, glass slides have been coated with special adhesives. The indirect immunofluorescence principle was applied: incubation in unlabeled primary antibody is followed by incubation in fluorescein (FITC) labeled secondary antibodies.