[The effect of fixation, dehydration and polymethacrylate imbedding on the results of immuno- and enzymehistochemical studies on lymphatic tissue and bone marrow]

Abstract

The influence of formaldehyde fixation, dehydration and polymethacrylate embedding on the results of immunofluorescence staining of immunoglobulin and enzymehistochemical demonstration of hydrolases, especially acid phosphatase and alpha-naphthylacetate esterase in human tonsils and bone marrow were studied. The best results were achieved by using 2% formaldehyde and 5% sucrose in 0.02 M phosphate buffer (ph 7.4) for fixation and ethyleneglycol monobutylether for dehydration. The procedures were carried out at 4 degrees C. For tissue embedding we used the commercially available polymethacrylate Kalloplast R. The polymerisation was carried out at -20 degrees C for 18 hours. This method permits a good preservation of morphological details and the survival of antigenic determinants and enzyme activity. Trypsin, pepsin and alpha-chymotrypsin were used to "unmask" the antigenic determinants in methacrylate sections. Trypsin and alpha-chymotrypsin revealed comparable results, but because of better practicability we used alpha-chymotrypsin. Using the described fixation, dehydration and embedding procedures it was possible to demonstrate both intracellular immunoglobulins (gamma, alpha and mu heavy chains; kappa and lambda light chains) and surface-bound immunoglobulins (mu and delta heavy chains; kappa and lambda light chains). Comparable results were achieved in human bone marrow biopsies. The results of histochemical demonstration of both enzymes were better in bone marrow sections than in that of the tonsils. We think, that the presented method is suitable in the diagnosis of myelo- and lymphoproliferative disorders on both the bone marrow and lymphatic tissue.