Methods in electron microscopic quantitative immunocytochemistry using adenohypophysis as a model

Sammanfattning: Quantitative ultrastructural immunocytochemistry (QUIC) is based on the assessment of immunogold labeling over the surface of ultrathin resin-embedded sections. The density of specific labeling, expressed as the number of gold particles/mm2 of a specific area can only be used as an approximative estimation of antigen concentration in the object.The reliability of QUIC depends on the quality of the methods used to prepare the samples; therefore, to make QUIC reliable, the entire preparative protocol! has to be evaluated, step by step. Secretory granules in cells of the pituitary gland have been chosen as a model system. They constitute targets that are sensitive enough for analyses of exogenous factors, such as fixation, dehydration, embedding and immunostaining. Morphological parameters, such as diameter, area, roundness and gray values could be measured by computerized image analysis and the results related to the experimental factors.Certain combinations of processing for EM were found to give the highest immunolabeling. A further criteria was, however, added-reproducibility of immunostaining among series of ultrathin sections. The most consistent labeling densities were found after cryofixation followed by physical dehydration and embedding in epoxy resin. One of the explanations could be that the greater resin surface relief of the latter provides a larger accessible area for the attachment of immunogold reactants. The advantages of certain techniques, such as EM in STEM mode and X-ray microanalysis were demonstrated and facilitated the evaluation of techniques in QUIC.