Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Effect of collidine (2,4,6-trimethylpyridine) on rat pineal gland and vas deferens nerves

Summary In previous work of our laboratory it was demonstrated that collidine (2,4,6-trimethylpyridine) abolishes the core osmiophilia and chromaffin reaction from rat pineal gland and vas deferens nerves. This abolition was apparent when tissues were briefly incubated in collidine or when they were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer substance. These and other results strongly suggested that the histochemical effect of collidine was due to depletion of monoamines stored in the vesicles core. To examine this hypothesis we studied in this work the effect of collidine on tissues that have taken up tritiated noradrenaline. It was found that tritium was released very rapidly to the incubation medium when collidine was applied to fresh tissues. This effect was not observed with other commonly used buffers such as cacodylate or phosphate. It was also found that tritium release also occurred, although to a lesser extent, when tissues were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer, and this release was not significant when collidine was applied to previously fixed tissues. Paper chromatographic analysis showed that the radioactive compound(s) extracted from tissues by collidine corresponded to noradrenaline and/or closely related compounds. An abstract of this work was sent to the 17th Annual Meeting of the Society for Neuroscience, New Orleans, Nov 16–21, 1987. Tomsig J.L. and Pellegrino de Iraldi A. Abstract 369-11.     

Loss of antibody binding to prefixed cells: Fixation parameters for immunocytochemistry

Summary The denaturing effects of various types of fixative solutions on 5 cell surface antigens on mouse T-lymphocytes (Thy-1, T-200, Lyt-1, Lyt-2, and Th-B) were studied. For this purpose, cells were fixed with paraformaldehyde, glutaraldehyde, acrolein and osmium tetroxide at various concentrations. Fixed cells were then incubated with monoclonal antibodies and appropriate second stage antibodies or conjugates. The degree of antibody binding to these cells was determined quantitatively using flow-cytometry with a fluorescence-activated cell sorter or with a semi-automatic micro-ELISA system. The data obtained indicate that paraformaldehyde and glutaraldehyde preserve all five tested antigen molecules, whereas antibody binding to cells fixed in acrolein and osmium tetroxide is rapidly reduced at increasing concentrations of the fixative. The optimal concentration of paraformaldehyde is in the range 0.5–1%, whereas glutaraldehyde should be used at concentrations between. 0.05 and 0.1%. Cells fixed with 0.5% paraformaldehyde or with 0.05% glutaraldehyde are stable and can be stored for at least one week prior to incubation with antibodies.     

The secretory ameloblast of the mini-pig foetus

Electron microscopic investigations of secretory ameloblasts from deciduous tooth germs of mini-pig foetuses and investigations of the ability of various fixatives to preserve these cells in tooth germs immersion-fixed in toto 5 min, 10 min, 15 min, 20 min and 40 min after death of the mother gave the following results:

Effect of collidine (2,4,6-trimethylpyridine) on rat pineal gland and vas deferens nerves. Further evidence for a monoamine-releasing effect

In previous work of our laboratory it was demonstrated that collidine (2,4,6-trimethylpyridine) abolishes the core osmiophilia and chromaffin reaction from rat pinal gland and vas deferens nerves. This abolition was apparent when tissues were briefly incubated in collidine or when they wer fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer substance. These and other results strongly suggested that the histochemical effect of collidine was due to depletion of monoamines stored in the vesicles core. To examine this hypothesis we studied in this work the effect of collidine on tissues that have taken up tritiated noradrenaline. It was found that tritium was released very rapidly to the incubation medium when collidine was applied to fresh tissues. This effect was not observed with other commonly used buffers such as cacodylate or phosphate. It was also found that tritium release also occurred, although to a lesser extent, when tissues were fixed in glutaraldehyde or osmium tetroxide using collidine as a buffer, and this release was not significant when collidine was applied to previously fixed tissues. Paper chromatographic analysis showed that the radioactive compound(s) extracted from tissues by collidine corresponded to noradrenaline and/or closely related compounds. An abstract of this work was sent to the 17th Annual Meeting of the Society for Neuroscience, New Orleans, Nov 16-21, 1987. Tomsig J.L. and Pellegrino de Iraldi A. Abstract 369-11.     

Ultrastructure and location of cytoplasmic fibrils inSpiroplasma floricola OBMG

The ultrastructure ofSpiroplasma floricola OBMG was investigated to identify subcellular structures that might be involved in motility and helicity. Optimal preservation for thin sectioning was achieved with either glutaraldehyde or a mixture of glutaraldehyde plus paraformaldehyde followed by OsO₄ and uranyl acetate. In thin sections, a 94-nm-wide band consisting of 4-nm-diameter fibrils was observed apposed to the cytoplasmic side of the plasma membrane. The band of fibrils extended axially the entire length of the cell. The addition of rethenium red to fixative solutions resulted in condensation of the fibrils. Freeze-substitution increased the apparent thickness of membranes but did not improve preservation of the fibrils. Freeze-fracturing revealed a 99-nm-wide zone containing few particles in fractured membrane surfaces. Treatment with deoxycholate or Triton X-100 to dissolve membranes yielded bands of fibrils comparable to those seen in thin sections. Based on these findings, a model indicating the intracellular location of the fibrils is proposed.     

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO₄ fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.Key words: frozen biopsy, electron microscopy, fixation, immunocytochemistry, skeletal muscle.     

Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves

Summary Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is elminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.     

Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves

Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is eliminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

Postembedding immunogold labeling for electron microscopy using "LR White" resin

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.     

Acrolein as a fixative for enzyme cytochemistry.

Since acrolein can penetrate more quickly and deeply into tissue blocks than glutaraldehyde, the possibility of the use of this aldehyde as a prefixative in enzyme cytochemistry was reinvestigated. At low concentrations, acrolein preserves the activities of the enzymes investigated, including those of glucose-6-phosphatase, which is known as one of the most vulnerable to aldehyde fixation; thus, acrolein is usable in enzyme ultracytochemistry. Enzyme activities are also preserved in tissues fixed with acrolein and glutaraldehyde combined. The rapid penetration of acrolein enables fixation in larger tissue blocks and provides greater freedom in specimen selection, especially important advantages when encountering heterogeneous materials as in pathology.     

Visualisation of messenger RNA directing peptide synthesis by in situ hybridisation using a novel single-stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity

Recently (Pecci Saavedra et al. 1982; Brusco et al. 1982, 1983) we have showed that the actual specificity of the rabbit anti-5-HT antibodies, is for the beta-carboline derivatives of 5-HT as a result of cyclization of the lateral chain. We explained this as resulting from the use of formaldehyde which acted both as a fixative in the preparation of the tissues, and as the coupling agent in the preparation of the immunogen. Following this line we have fixed several brain stem specimens with 0.5% p-benzoquinone; 3% glutaraldehyde; 4% paraformaldehyde plus 0.25% glutaraldehyde and compare the results with tissues fixed in 4% paraformaldehyde. Glutaraldehyde and p-benzoquinone do not produce cyclization of 5-HT but immobilize monoamines in situ. As expected, the antibodies applied according to the PAP technique did not stain the neuronal bodies of the raphe system, known to contain 5-HT when 3-4% glutaraldehyde or 0.5% p-benzoquinone were used. Good staining was obtained with 4% paraformaldehyde alone or with 4% paraformaldehyde plus 0.25% glutaraldehyde. A quantitative assay of the spot test of Larsson (1981) was devised for measuring in vitro the inhibitory effects of 5-HT, of the 5-HT-BSA complex and of the cyclic derivative, 6-OH-1,2,3,4-tetrahydro-beta-carboline. The results confirmed that the avidity of the antiserum is much greater for the cyclic derivatives contained in the 5-HT-BSA complex and for 6-OH-1,2,3,4-tetrahydro-beta-carboline than for 5-HT. It is concluded that the formation of a new ring by the lateral chain of 5-HT is responsible of the in-vitro and in the tissue immunoreactivity of the anti-5-HT-antibodies.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Parathyroid cell variants may be provoked during immersion fixation

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Factors affecting lead capture methods for the fine localization of rat lung acid phosphatase

Synopsis Gomori's lead capture method for acid phosphatase localization was adapted for the electron microscope by Holt & Hicks (1961a). The method gave good results in rat liver, but poor tissue preservation with no reaction product in rat lung, and was, therefore, investigated in order to find the optimum conditions for the ultrastructural localization of rat lung acid phosphatase. The conditions investigated included the use of glutaraldehyde or depolymerized paraformaldehyde as the fixative, with and without dimethylsulphoxide; the effect of freezing the tissue; the pH of the incubation medium; and the use of glycerophosphate, naphthol AS-BI phosphate or -naphthyl phosphate as substrates. Improved preservation of ultrastructure with increased yield of reaction product was obtained by prefixing lung in glutaraldehyde containing 10% dimethylsulphoxide, freezing the tissue and incubating at pH 5.7 with -naphthyl phosphate. Tissue preservation was acceptable and dense deposits of reaction product occurred in lysosomal elements of all the alveolar cells and especially in macrophages. Deposits were also found closely associated with the lamellae of the inclusion bodies of Type II cells.     

Immunofluorescence localisation of microtubules in plant root tips embedded in butyl-methyl methacrylate

Most immunofluorescence studies of microtubules in plant cells have used enzymatically isolated cells whose position in the organ and stage of development was unknown. Moreover, attempts to label microtubules in cells in intact tissue have suffered from poor resolution and inferior tissue preservation. To overcome these difficulties, I have used a removable embedding resin to localise microtubules in situ. Tissues were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde and embedded in butyl-methyl methacrylate. Semi-thin sections were extracted with acetone to remove the resin and labelled with anti-tubulin followed FITC-labelled second antibody. Brightly stained microtubule arrays were clearly visualized. This reversible embedding medium should provide a powerful tool to study developmental changes in microtubule arrays in growing and differentiating plant tissues.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Cytochemical demonstration of adenylate cyclase in cardiac muscle: effect of dimethyl sulfoxide.

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.