Lysozyme antigenicity and tissue fixation.

The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues aqueous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.     

The antigenicity of regenerating tail tissue in the newt Diemictylus viridescens

Young adult male rabbits were inoculated with antigens prepared from regenerating (blastema stage) and nonregenerating tail tissues of the newtDiemictylus viridescens. Blood was collected from these rabbits after six weeks of semiweekly injection, two weeks of respite, and two more weeks of injections. A Freund adjuvant was added to the antigen preparations at the time of injection in order to elicit the anamnestic effect.Ouchterlony agar diffusions of the newt antigen preparations vs. the rabbit antisera were carried out. The resulting patterns of precipitation bands were compared and photographed.The strongest precipitation reactions of a given series were those between the antigen preparations made from nonregenerating tissue and their homologous antisera. The weakest reactions occurred between regenerating tissue antigens and regenerating tissue antisera. The strength of the antigen-antibody reactions was judged by the number of bands appearing in the diffusion plate and by the distinctness of these bands. Reactions of intermediate strength occurred between regenerating antigens and nonregenerating antisera, between nonregenerating antigens and regenerating antisera, and between antigens and antisera of different series.The loss of antigenicity during the blastemal period was considered to be related to the destruction of tissue in the wound areas at this time, and to indicate a quantitative rather than a qualitative loss of protein in regenerating tissue.This work is taken from data submitted to the Department of Biology of Fordham University in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and was supported in part by a grant from the New York City Cancer Committee of the American Cancer Society.The author acknowledges his indebtedness to Dr.Alexander Wolsky of Fordham University, under whose direction this investigation was carried out, and Dr.John J. Corbett of Manhattan College and Lederle Laboratories.     

Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears

Summary Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic intergrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2–10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.     

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry

The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3'-diaminobenzidine (DAB). In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane. In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.     

Cell and tissue fixation, 1972-1982

Conclusions There have been changes in the practice of fixation over the past 10 years. There seem to be at least two different pressures working. On the one hand, there is increasing diversity in cell biological techniques which in turn demands more variable fixation procedures. Some of these have been outlined. Some of this change in practice has percolated through to pathology where it has been found to be diagnostically useful. In surgical pathology on the other hand, there is the continuing financial pressure for more rapid through-put of specimens which includes more rapid fixation, often with the loss of potential for subsequent chemical investigations. These are the horns of the dilemma; both are wanted at the same time.It seems that there is no magical fixative in sight which will permit all investigations on all tissues. Rather, it seems that the future will hold increasing diversity in fixation procedures which will demand that practitioners be well informed as to possibilities which, hopefully, may lend to better understanding of the problems and mechanisms of fixation.     

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry

Summary The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3-diaminobenzidine (DAB).In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane.In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.This study was supported by Grant No. 12X-537 from the Swedish Medical Research Council     

Special symposium: fixation and tissue processing models

Abstract

Fixation and processing of tissue to paraffin blocks permit thin (4-5 µm) sections of tissues to be cut. Tissues and their subcellular components and surrounding stroma are visualized by cutting thin sections and staining them histochemically or immunohistochemically and viewing the sections using a bright field microscope. During the last century, anatomists and pathologists have used fixation with 10% neutral buffered formalin (10% NBF) as the fixative of choice. Also, both human and veterinary pathologists have trained to use fixation with 10% NBF, so these professionals are reluctant to change the familiar microscopic appearance of diagnostic tissues by using different fixatives. In addition, the effects of tissue processing on the microscopic appearance of tissue essentially has been ignored in most studies. Archives of paraffin blocks of pathological tissue contain essentially paraffin blocks fixed in 10% NBF. Therefore, if retrospective studies use archival paraffin blocks to correlate the molecular features of diseases with their outcomes, the studies must be based on tissue fixed in 10% NBF. Studies of how fixation in 10% NBF interacts with histochemical and immunohistochemical staining are limited in number and most are based on relatively long fixation times (≥36 h). Currently, fixation times in 10% NBF have been reduced to <24 h. Little is known about fixation in 10% NBF and its interaction with tissue processing for any period of fixation, especially short times. Less is known about how fixation of tissues with 10% NBF interacts with more modern assays using immunohistochemistry, real time quantitative polymerise chain reaction (PCR), and techniques that depend on analysis of proteins extracted from paraffin blocks including multiplex immunoassays or mass spectrometry. In general, multiple antibody–antigen combinations are reported not to work in tissues fixed in 10% NBF, i.e., loss of immunorecognition is nearly complete for such antibody–antigen combinations as Ki67/MIB, estrogen receptor alpha (ERα) and Progesterone receptor (PR), and partial for Bcl-2. Several models have been developed to study the interactions of tissue fixation and immunorecognition, but most have viewed the problem with immunorecognition as completely caused by fixation. Also, some of the models discussed in this special symposium do not predict the effects of fixation on frozen tissues fixed in 10% NBF and not processed to paraffin blocks. This article is a brief review of issues attending the use of 10% NBF combined with tissue processing as an interrelated process to study biomarkers identified by immunohistochemistry.     

Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears.

Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.     

Histological assessment of PAXgene tissue fixation and stabilization reagents

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Preservation of tissue mucins by freeze-drying and vapour fixation

Summary Histochemical studies previously undertaken showed that tissue mucins (glycoproteins) of the rat submaxillary salivary gland and other organs are preserved very satisfactorily by formaldehyde vapour treatment applied after freeze-drying of the tissue.It was thought desirable to confirm these histochemical findings by quantitative chemical data. This was performed by studying the effect of formaldehyde vapour treatment on the solubility of proteins in the freeze-dried rat submaxillary gland.Large quantities of protein (about 30 to 60 per cent of the dry weight) could be removed by aqueous extraction from the freeze-dried control samples, which had not received any formaldehyde vapour treatment, but very little protein (about 0.5 to 4 per cent of the dry weight) could be extracted from those samples which had been exposed to this vapour at 50°C for 3 hours.Each of the experiments performed confirmed this overall picture, but there were differences in the amount of protein extracted among the control samples, as well as among the formaldehyde vapour treated ones; it has been suggested that these differences were due to variations in the proportion and/or type of protein present, most probably caused by fluctuations in the content of secretory mucins.In part fulfilment for the Doctorate of Philosophy, University of London.