TECHNIQUES IN IMMUNE-ELECTRON MICROSCOPY .1. CRYOSUBSTITUTION

Normal skin was cryoprotected by submerging it in a mixture of 30% dim ethylformamide (DMF) in PBS or RPMI. Subsequently it was frozen in liq uid propane gas. Cryosubstitution was carried out at -90 degrees C by using methanol to which uranyl acetate or osmium tetroxide were added. The tissue was embedded in either Lowicryl K4M at -40 degrees C or in Epon at +60 degrees C. The tissue was evaluated by its overall preser vation of ultrastructural details and by its labeling intensity after incubation with either anti-desmoglein or anti-type VII collagen monoc lonal antibodies. The mixture of DMF and PBS caused an electron-dense precipitate within the cell. The overall morphology was better in Epon -embedded material than in K4M-embedded material. However, the labelin g was best in K4M material. Regardless of whether the tissue was embed ded in Epon or K4M, the addition of osmium tetroxide markedly reduced the degree of labeling. (C) Munksgaard, 1994.