title: Use of light microscopic immunotechniques in selecting preparation conditions and immunoprobes for ultrastructural immunolabelling of lactoferrin.
abstract: Successful postembedding immunolabelling for electron microscopy is sometimes difficult to achieve. We propose that light microscopy can be used (1) to detect quickly processing steps which have an adverse effect on the tissue antigenicity and (2) to check the specific reactivity of the immunogold detecting system normally employed at the ultrastructural level. The individual steps of fixation, dehydration and embedding were tested for their ability to preserve antigenicity by light microscopic peroxidase--anti-peroxidase cytochemistry. Steps that severely reduced antigenicity were replaced by less destructive alternatives compatible with reasonable ultrastructural preservation. The specific reactivity of the immunogold detecting system was assessed by using the light microscopic immunogold-silver staining method. We studied the antigen lactoferrin in human neutrophilic granulocytes from patients with chronic myeloid leukaemia. We obtained strong immunolabelling of specific granules and good ultrastructural preservation using routine methods at room temperature. For lactoferrin the method of choice was to fix in 3% paraformaldehyde/0.1% glutaraldehyde followed by 1% OsO4, dehydrate in 70% ethanol, embed in LR White resin and polymerize at 40 degrees C for 40 h. These conditions may not be suitable for all antigens and we emphasize that for each new antigen a similar study should be carried out.
authors.affiliation: Department of Haematological Medicine, University of Cambridge, UK.
authors.forename: H C
authors.initials: HC
authors.lastname: Mutasa
authors.forename: E C
authors.initials: EC
authors.lastname: Pearson
mesh_terms: D005404:Fixatives; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007781:Lactoferrin / Q000276:immunology / Q000378:metabolism*; D007782:Lactoglobulins / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D012116:Resins, Plant / Q000494:pharmacology
chemical_list: D005404:Fixatives; D007782:Lactoglobulins; D012116:Resins, Plant; D007781:Lactoferrin