Paraformaldehyde Fixation Research Articles

The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity

The rat monoclonal antibody YC5/45 has been shown to react specifically with serotonin containing neurones, and does not detect dopamine containing sites. On the other hand the monoclonal antibody recognizes dopamine, since it is capable of inhibiting the binding of 3H-YC5/45 to sheep red blood cells coated with serotonin albumin conjugate, or the direct agglutination of the same cells by the monoclonal antibody. Tryptamine and 5-methoxytryptamine are even better inhibitors, while derivatives with a modified-CH 2-CH 2-NH 2 group are inactive. Treatment of the active compounds with paraformaldehyde increases their efficiency dramatically. The binding of antibody was also tested by cross linking with paraformaldehyde: dopamine, serotonin. tryptamine. 5-methoxytryptamine etc., to serum albumin. Only the serotonin derivative was active. The result shows that the discrimination specificity observed in immunohistochemistry is due to the paraformaldehyde fixation. It is concluded that for histochemical detection of serotonin by YC5/45, two reactive sites are necessary. The -CH 2-CH 2-NH 2 group is probably modified by formaldehyde to produce a cyclic derivative to form the antibody recognition site. A different formaldehyde reactive site is required for cross linking to the tissue and this is provided by the presence of the 5-OH of serotonin which is absent in the others.

Immunoelectron microscopic localization of immunoglobulins and complement in human renal glomeruli.

A study was undertaken to evaluate some processing variables affecting the immunoelectron microscopic demonstration of immunoglobulins and complement (C3) in human glomeruli. Percutaneous biopsies were performed on 28 patients with various types of glomerulonephritis. Light microscopic, electron microscopic, and immunofluorescence examinations were performed by routine methods. For immunoelectron microscopy, fixation in paraformaldehyde (PA) or periodate-lysine-paraformaldehyde (PLP) was used. With the diffusion technique, using tissue chopper or cryostat sections, human immunoglobulin (Ig)G, IgA, IgM, and C3 were localized in glomeruli with peroxidase-labeled antisera. Using PLP and the tissue chopper sections, good ultrastructure was achieved. The antigens could be demonstrated in intramembranous, subepithelial, subendothelial, or mesangial immune deposits. Penetration of antibodies and quality of peroxidase reaction in the cryostat sections did not differ from that of the tissue chopper sections. Freezing and thawing, however, resulted in inferior morphology. If PA was used, the antigens could not be reliably demonstrated. The results of light microscopy, electron microscopy, and immunofluorescence microscopy were in good agreement with those from the immunoperoxidase procedure. The present study shows that PLP preserves well the antigenicity of human immunoglobulins and C3, resulting in good ultrastructure. PA fixation, on the contrary, caused a loss of antigenicity before an adequate ultrastructure could be achieved.

Immunohistochemical identification of mastocytes

Antibodies to mastocytes were localized in cells and tissues following formaldehyde or paraformaldehyde fixation and embedding into paraffin or Durcupan ACM, and after chymotrypsin treatment of the sections. Immunomicroscopic examination yielded a more distinct picture of the amount and localization of mastocytes in different tissues.

Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis

The objective of this study was to find a simple method to preserve cells for subsequent flow cytometry (FACS) analysis. We determined that fixation in 1% paraformaldehyde—0.85% saline solution did not significantly alter the Coulter volume, light scatter or fluorescence properties of the cells. This method was suitable for all cell types analyzed, including mouse, human and rat lymphoid cells, erythrocytes and transformed cell lines. Furthermore, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4°C in the dark for at least a week prior to FACS analysis. This method should prove useful to those who work with in vivo derived specimens or have limited access to flow cytometry facilities.

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.

Modification of the Platelet Suspension Immunofluorescence Test

This study reports two modifications of the Platelet Suspension Immunofluorescence Test which make it more convenient to use for the rapid identification of platelet-specific antibodies and for screening individuals if platelet donors of a particular type are urgently required. Platelets frozen both before and after paraformaldehyde (PFA) fixation were compared with fresh PFA-fixed platelets using anti-PLAl and anti-HLA antisera. Further, a comparison was made between the results of the test when performed in tissue culture trays or in tubes as originally described. Platelets, prefixed with PFA and then frozen appeared similar in all respects to fresh PFA-fixed platelets with no loss of antigenicity and no non-specific fluorescence. Although platelets fixed after frozen storage were also satisfactory, they were less convenient to use and lost some brilliance in staining. When the test was performed in tissue culture trays, there was no loss in sensitivity, and the volume of antiserum used was halved. However, the main advantage was the efficiency and ease with which the test could be performed, especially when handling large numbers of samples.

Ultrastructural Immunocytochemistry of Five Rat Pituitary Adenomas

Conventional light and electron microscopic techniques failed to clarify the cellular composition and derivation of spontaneous and induced, intrasellar and transplanted pituitary adenomas in rats (1). In the present work, electron microscopic immunocytochemistry was applied to evaluate five adenohypo-physial tumors using a technique described by Moriarty and Garner (2). Spontaneously occurring pituitary adenomas (group 1) were harvested from aging female Long-Evans rats. R-Amsterdam rats were treated with 2 x 1.0 mg estrone acetate (HogivaI) s.c. weekly for 6 months. Pituitary adenomas in excess of 30 mg were removed from these animals to make up the tumors of group 2. Groups 3 and 4 consisted of estrogen-induced autonomous transplan¬ted pituitary tumors MtT.WlO and MtT.F4. Group 5 was a radiation-induced transplanted autonomous pituitary tumor MtT.W5. The tumors of groups 3,4 and 5 were allowed to proliferate in host rats 6-8 weeks prior to removal for processing. Tissue was processed for transmission electron microscopy (glutaraldehyde fixation, OsO4 postfixation and epoxy resin embedding), and electron microscopic immunocytochemistry (3% paraformaldehyde fixation and Araldite embedding).

Immunohistochemical identification of immunoglobulin A on ultrathin tissue sections.

Antigenic determinants of the heavy chain of immunoglobulin A (IgA) were identified within immunocytes by immunohistochemical techniques applied to ultrathin sections of rat ileal mucosa embedded in eposy resin. Successful localization of IgA depended upon the utilization of chemical fixatives containing paraformaldehyde or physical fixation employing a freeze-drying technique. Partial removal of the epoxy resin from the ultrathin tissue section with metallic alkoxide solutions or pretreatment with H2O2 was essential prior to the sequential application of the immunological reagents. IgA was associated with the rough endoplasmic reticulum (RER) and nuclear regions of immunocytes. These results are similar to those of other investigators employing immunochemical localization before embedment.

Distribution of an endogenous lectin during myogenesis as measured by antilectin antibodies

Antibodies to electrolectin, a lectin endogenous to embryonic skeletal muscle, have been used to study the distribution of lectin during myogenesis in L 6 cells by using immunohistofluorescence microscopy. Evidence for both surface and cytoplasmic forms of lectin is presented. Cytoplasmic lectin does not appear to be strongly regulated by cell cycle or differentiation; it is uniformly distributed throughout the cytoplasm and all cells have about the same quantity as judged by the intensity of fluorescence. Surface lectin is characterized as being organized into puntate structures which can be found on both the upper and lower surfaces of the cell and occasionally on the surface of the dish. Surface lectin is strongly influenced by the differentiation of cells, i.e. it is highest on mononucleated cells and low on myotubes. Paraformaldehyde fixation causes patches of lectin to develop which are not found when live cells are used. The effect of paraformaldehyde is accentuated by Ca 2+.

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.

Distribution and mobility of specific antigens on isolated guinea pig epidermal cells

Surface distribution and mobility of epidermis-specific antigens were studied under various conditions in trypsinized, isolated guinea pig epidermal cells by means of immunoferritin electron microscopy. Fresh or paraformaldehyde-fixed cells were treated with rabbit antiserum specific for epidermal cell surface antigens of the guinea pig at 4°C and then labeled with ferritin-conjugated sheep anti-rabbit IgG at 4° or 37°C for 15 min, respectively. In fresh cells labeled at 4°C ferritin was bound uniformly over the entire cell surface in all keratinocytes with some tendency to form small aggregates. In fresh cells labeled at 37°C there were large aggregates of ferritin that were separated by long unlabeled regions (patch formation) and pinocytosis of ferritin in all basal and lower spinous cells. Some upper spinous cells also showed formation of small clusters and pinocytosis of ferritin. Other upper spinous and granular cells were uniformly labeled on the entire cell surface and showed no evidence of pinocytosis. The surface antigens of isolated epidermal cells were demonstrated to be mobile in the membrane plane of basal and lower spinous cells but immobile within the membrane of granular cells. Immobilization of the antigens seemed to occur first in upper spinous cells. It is possible that decrease in fluidity is one of the qualitative changes of the epidermal cell membrane associated with keratinization. Fixed basal and spinous cells showed many small aggregates of ferritin while fixed granular cells displayed uniform labeling. It was suggested that modification of the antigenicity due to paraformaldehyde fixation resulted in the patchy appearance of labeling in lower keratinocytes.

A Simple Immunofluorescence Test for the Detection of Platelet Antibodies

Immunofluorescence tests on platelets have always been hampered by nonspecific fluorescence caused by non-immunological binding of plasma proteins to the platelet membrane. It was found that this could be easily overcome by fixation of the cells with paraformaldehyde (PFA). By using PFA-fixed platelets, a simple method for the detection of platelet antibodies, the platelet suspension immunofluorescence test (PSIFT) was developed. PFA fixation did not alter or inactivate the platelet antigens tested. Platelet-reactive antibodies detected specifically with the PSIFT included platelet-specific agglutinins of the IgM class, non-agglutinating platelet-specific antibodies of the IgG class, drug-dependent platelet antibodies, HLA antibodies, as well as anti-A and anti-B antibodies. The sensitivity of the new test was satisfactory, as was its reproducibility. Measurement of platelet immunofluorescence was possible in a continuous flow microfluorometer, making a principle, quantitation of platelet antibodies and antigens possible.

Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.

1. Myotomal muscle cells from embryos of Xenopus laevis were cultured as a monolayer either alone or together with neural tube cells from the same embryos. 2. Spontaneous twitching and contractions evoked by electrical stimulation of neural perikarya were observed only in nerve-contacted muscle cells, and could be abolished by curare or alpha-bungarotoxin. 3. Within 2 days in culture muscle cells not contacted by nerve developed one or more discrete patches of acetylcholine (ACh) receptors as revealed by staining with fluorescent conjugates of alpha-bungarotoxin. Similar patches were also seen when staining was carried out after paraformaldehyde fixation, suggesting that they were not induced by the dyetoxin conjugate. 4. Radioautography after labelling with [125I]alpha-bungarotoxin revealed patches with grain densities approximately twenty-five-old greater than over the remainder of the cell. 5. Fluorescent stain on innervated cells was restricted to the path of nerve-muscle contact and sometimes extended for greater lengths than the largest patches seen on non-contacted muscle cells. 6. Similar long bands of stain associated with nerve--muscle contacts were observed when cultures were grown in high concentrations of curare and carbachol which prevented spontaneous twitching. They were also seen in cultures in which the addition of neural tube cells was delayed for 2-3 days. 7. It is concluded that innervation caused receptors to accumulate at sites of nerve-muscle contact and that this process can operate independently of muscle activity.

Variations in kidney ultrastructure with a variety of paraformaldehyde fixation techniques.

Variations in fine structure of glomeruli and proximal tubules were studied to determine the results of several fixation techniques with paraformaldehyde. Immersion fixation techniques generally resulted in poor fixation, while the quality of fixation by perfusion was directly related to the concentration of the paraformaldehyde. The best technique was fixation by perfusion and immersion in 3% paraformaldehyde for 2 h followed by a post-fixation in 1% OsO4 for 1 h. Intracellular edema was noted in all epithelial, mesangial and proximal tubular cells that were from poorly fixed kidneys. Swollen mitochondria and the dilated endoplasmic reticulum were common in many of those cells. Mesangial and proximal tubular cells were the first to swell. Poor fixation produced the early stages of autolysis. The criteria for good paraformaldehyde fixation are discussed.

Polar appearance and nonligand induced spreading of measles virus hemagglutinin at the surface of chronically infected cells

Fixation with glutaraldehyde (GA) and paraformaldehyde (PFA) preserved measles virus hemagglutinin at the surface of chronically infected cells. Cells fixed with PFA but not with GA exhibited hemadsorption with green monkey cells. PFA fixation, in contrast to GA fixation, also preserved the immunogenicity of measles virus hemolysin. These fixatives and the removal of the measles virus hemagglutinin from the cell surface by trypsin enabled studies of the appearance of the hemagglutinin at the surface membrane. Results obtained by immunofluorescence technique and by hemadsorption indicated that measles virus hemagglutinin appeared polarly at the cell membrane and then spread around the surface. This was substantiated by measurements of the immunofluorescence intensity at the single cell level per membrane unit and per cell, and by measuring the binding of iodinated immunoglobulins per 10 6 cells. The appearance was inhibited by sodium azide and cytochalasin B. The spreading was not inhibited by sodium azide, but was influenced by cytochalasin B. The spreading did not proceed at 4°C. On the basis of these findings, a hypothetical model for appearance and spreading of measles virus hemagglutinin was proposed.

Paraformaldehyde fixation in immunofluorescence and immunoelectron microscopy: Preservation of tissue and cell surface membrane antigens

By means of the immunofluorescence technique and immunoelectron microscopy it is demonstrated that fixation of tissues and cells with 1% paraformaldehyde does not seriously affect tissue and membrane-bound antigens. Higher concentrations (2% and 4%) of paraformaldehyde and addition of glutaraldehyde in a concentration of over 0.1% to 1% paraformaldehyde caused a rapid loss of antigenicity. Addition of glutaraldehyde in very low concentrations (0.01–0.05%) improved the morphological conditions required for immunoelectron microscopical studies. Surface-bound antigens were immobilized by the fixation procedure with 1% paraformaldehyde, which abolished or greatly reduced the capping process. Finally cells incubated with labelled antisera after fixation could be kept for about two weeks before screening.

Immobilization of membrane H-2 antigens by paraformaldehyde fixation.

Immobilization of membrane H-2 antigens by paraformaldehyde fixation.

Cytological localization of Dengue-2 antigens: an immunological study with ultrastructural correlation.

Immunofluorescence and electron microscopy were used to study the development and cytological distribution of dengue-2 virion and nonvirion antigens in monkey kidney cells. The type and combination of fixatives were found to affect the intensity of fluorescence. Paraformaldehyde fixation alone resulted in a low level of fluorescence, but an additional fixation step with ethanol or acetone resulted in maximum staining. No fluorescence was obtained after fixation with glutaraldehyde alone or in combination with other solvents. Methanol caused selective ablation of fluorescence by antibody to purified virions. Dengue-specific fluorescence that was most intense in the perinuclear area radiated in a granular pattern of decreasing intensity into the cytoplasm. The perinuclear fluorescence was associated with nonvirion antigens and the cytoplasmic fluorescence was associated with virion antigens. Electron micrographs of infected cells revealed vesicular bodies with reticular electron-dense centers, clusters of structurally complete virions deep within the endoplasmic reticulum, and single virus particles in vacuoles near the periphery of the cell.

Paraformaldehyde fixation of mouse cells with preservation of antibody-binding by the H-2 locus.

We have studied the antibody binding capacity of mouse H–2 antigens after fixation with paraformaldehyde. Spleen cells and erythrocyte stroma fixed for one hour in 0.9% paraformaldehyde were found to bind anti‐H–2 antibodies to essentially the same extent as fresh material. Mouse macrophages fixed in this way were shown to be unable to pinocytose peroxidase. The fixation procedure described here should be useful in studies of the amount and distribution of H–2 antigens on mouse cells.

The termination of callosal fibers in the paravisual cortex of the rat

Summary A light and electron microscope study of the callosal connections of the paravisual cortex of the rat has shown that the callosal axons entering this region terminate in all layers but with highest density in deeper layer I, throughout layer II, and in the upper half of layer III. The callosal endings contain round vesicles (following para-formaldehyde fixation) and form asymmetric contacts with dendritic branches and spines. In addition callosal endings contact non-pyramidal cell somata and their major dendritic trunks. Callosal terminals never contact pyramidal cell somata or their proximal axon segments and only extremely rarely do they contact their major dendritic trunks. This is consistent with the finding that with the paraformaldehyde fixation used in this study axo-somatic synapses and synapses onto proximal axon segments of the pyramidal cells of this region are formed by synapses containing flattened vesicles. While filamentous terminals and correlated neurofibrillar rings have been found occasionally, there is no evidence of a filamentous degenerative reaction of the callosal terminals. These most frequently undergo a dense degenerative reaction which is most evident from 3 to 7 days after a contralateral occipital lesion.