Premium
Techniques in immuno‐electron microscopy
Author(s) -
SchaumburgLever G.,
Fehrenbacher B.,
Möller H.,
Nau P.
Publication year - 1994
Publication title -
journal of cutaneous pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.597
H-Index - 75
eISSN - 1600-0560
pISSN - 0303-6987
DOI - 10.1111/j.1600-0560.1994.tb00708.x
Subject(s) - osmium tetroxide , uranyl acetate , osmium , dimethylformamide , chemistry , electron microscope , incubation , methanol , nuclear chemistry , chromatography , pathology , staining , biochemistry , organic chemistry , medicine , solvent , physics , optics , catalysis , ruthenium
Normal skin was cryoprotected by submerging it in a mixture of 30% dimethylformamide (DMF) in PBS or RPMI. Subsequently it was frozen in liquid propane gas. Cryosubstitution was carried out at −90°C by using methanol to which uranyl acetate or osmium tetroxide were added. The tissue was embedded in either Lowicryl K4M at −40°C or in Epon at +60°C. The‐ tissue was evaluated by its overall preservation of ultras‐tructural details and by its labeling intensity alter incubation with either anti‐clesmoglein or anti‐type VII collagen monoclonal 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‐embedclecl material. However, the labeling was best in K4M material. Regardless of whether the tissue was embedded in Epon or K4M, the addition of osmium tetroxide markedly reduced the degree of labeling.