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An appraisal of low‐temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies
Author(s) -
Robertson David,
Monaghan Paul,
Clarke Catherine,
Atherton Amanda J.
Publication year - 1992
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1992.tb03253.x
Subject(s) - antigenicity , membrane , electron microscope , suspension culture , cytoplasm , fixation (population genetics) , biology , pathology , biomedical engineering , microbiology and biotechnology , biophysics , cell culture , antigen , biochemistry , immunology , medicine , physics , genetics , gene , optics
SUMMARY The progressive lowering of temperature (PLT) method of embedding for electron microscope immunolabelling has been examined with the objective of formulating a standardized protocol which can be applied to a wide variety of samples. The methods described cover fixation, processing of samples by the PLT method, embedding in Lowicryl HM20 and subsequent immunolabelling. Each of the steps in the fixation and embedding protocol has been assessed for its potential to retain both morphology and antigenicity. Comparison of samples embedded in Lowicryl K4M and HM20 at −25°C indicate an increased membrane contrast in HM20 sections, and a further improvement in morphology when samples were embedded in HM20 at −50°C rather than at −25°C. The results of applying the methods described are demonstrated in a range of samples of both mammalian and botanical origin, which includes solid tissues, cells in suspension, and cells grown in vitro as a monolayer culture and embedded in situ . Samples processed by this method have been immunolabelled using a wide range of antibodies recognizing nuclear, cytoplasmic, cell membrane and extracellular matrix antigens.