Microwave fixation of marine invertebrates

Marine invertebrates may be rapidly fixed for histological examination using microwave irradiation generated by household microwave ovens. Ten-second irradiation of whole intact clams gave tissue fixation equal or superior to standard procedures using formaldehyde solutions and eliminated the need for that hazardous chemical. We suggest that invertebrates can be fixed while relaxed in sea-water baths, without having to remove or open the shell, and that invertebrates in bottom sediment cores also may be fixed in situ without being disturbed.     

Microwave fixation of rust-infected wheat leaves

Summary Wheat leaves infected with stem rust (Puccinia graminis tritici) were infiltrated with fixative, subjected to microwave irradiation, and sliced with a vibratome. The slices were probed with antibodies, lectins, or neoglycoproteins, and processed for electron microscopy, In tissue irradiated for 10 sec to 40°C, 45°C, or 50°C, the quality of structural preservation was indistinguishable from that in control tissue subjected to conventional fixation (3 h in fixative at room temperature). The best preservation of fungal antigenic cell surface material was achieved with 10 sec of microwave-induced heating to 45°C in the presence of fixative, followed by 10 min in fixative at room temperature. Under these conditions, twice as many antigenic sites were detected on the fungal surface than in non-irradiated (power-off control, or conventionally-fixed) tissue. The microwave fixation protocol with heating to 45°C was used in experiments to probe infected tissue with lectins or neoglycoproteins. Most of these probes had been labelled with biotin, and this label was detected with goat anti-biotin IgG and rabbit anti-goat IgG/gold. The gold markers were localized mainly at some distance outside the outer wall layer of hyphal cells, indirectly confirming the presence of unstained extramural material that had been detected in earlier work in freeze-substituted specimens. Of seven lectins, all with demonstrated ability to bind to cross sections of intercellular hyphal walls, only concanavalin A and wheat germ lectin bound to the fungal surface. Of four neoglycoproteins, -D-glucosyland -D-mannosyl-BSA bound to this surface, but only the binding of the glucosyl conjugate was inhibitable with hapten. We concluded that the surface composition of these cells is less complex than previously suggested from studies using post-embedding cytochemistry.Abbreviations BSA bovine serum albumin - ConA concanavalin A - IWF intercellular washing fluid - NC nitrocellulose - OD optical density - PBS phosphate-buffered saline - PEG polyethylene glycol - TBS Tris-buffered saline     

Microwave fixation of water-cooled insect tissues for immunohistochemistry

Summary In this paper a new technique for microwave-accelerated tissue fixation and washing is described. The temperature of the irradiated specimens is controlled by means of a specially designed water-cooling device. Two types of fixation fluids, one of them containing rather concentrated picric acid and a washing procedure were tested empirically on dissected nervous tissue of the Colorado potato beetle,Leptinotarsa decemlineata (Say). Entire beetles were processed in a similar way. It was shown that specimens could be irradiated as long as was required for good fixation and washing results, without the accumulation of excessive heat. No differences in morphology and immunoreactivity were observed when compared with standard immersion-fixed controls. A considerable reduction in processing time was achieved.     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

Microwave fixation provides rapid, primary fixation for light and electron microscopy and for immunohistochemistry and immunocytochemistry.

A relatively new approach to specimen preservation for morphologic studies uses microwave energy and chemicals. Microwave fixation can produce fixation results equal in quality to chemical fixation methods and equal in speed to freeze fixation methods. The importance of this microwave fixation technology lies in its potential to provide a standardized fixation approach in histopathology, immunohistochemistry, and immunocytochemistry.     

Microwave stabilization versus chemical fixation. A morphometric study in glycolmethacrylate- and paraffin-embedded tissue

Summary A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate).An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37–46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10–20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.     

Shrinkage of lung after chemical fixation for analysis of pulmonary structure-function relations

Glutaraldehyde is widely used to chemically fix lungs for analysis of pulmonary structure-function relations. Accurate interpretation of observations on fixed tissue requires a clear definition of any artifacts, such as tissue shrinkage, resulting from fixation with glutaraldehyde. Two experimental procedures were used in this study to examine possible shrinkage artifacts resulting from fixation of lung by glutaraldehyde. In the first, isolated perfused dog lungs were rapidly frozen at different transpulmonary pressures. Samples were then freeze substituted at -50 degrees C using 70% ethylene glycol with and without fixatives present. In the second series of experiments, the left lungs of mongrel dogs were fixed by vascular perfusion with glutaraldehyde at different transpulmonary pressures. In both series of experiments any changes in linear dimensions resulting from the fixation procedure were measured. Also, the presence of aldehyde was demonstrated by a positive reaction with Schiff reagent. The results demonstrate that lung tissue fixed either by vascular perfusion or freeze substitution tends to shrink to about the same extent. This shrinkage is reasonably constant at about 9% for transpulmonary pressures of 5 and 15 cmH2O and increases to about 15% when the transpulmonary pressure reaches 25 cmH20.     

Microwave fixation: Its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy

Summary Human tissues, both biopsy and postmortem, and tissues from rodents were fixed by microwaves at various temperatures and compared against formaldehyde-fixed material. Conventional stains, including trichromes, worked well. Red cell were lysed, but white cells were fixed, thus permitting diagnoses of various inflammatory states. Malignant cells were equally well-preserved by the two methods. Histochemical investigations of mucosubstances, lipids and various hydrolases showed no significant difference between the two techniques. Some neurological stains, however, were not as good following microwave treatment. Immunocytochemical localization of IgA, IgM and IgG showed no significant difference after microwave fixation compared to that in tissues fixed with formaldehyde. Microwave fixation did not lead to a greater tissue shrinkage than that obtained with formaldehyde fixation. Both were significantly less than that following treatment with phosphate-buffered saline alone. Electron microscopy gave results which were interpretable, but with damage resembling early postmortem change. Microwave fixation is complete in approximately 1–2 min.The mechanism of fixation appears to be due to denaturation associated with disulphide bond formation and a decrease in solubility of proteins.     

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy

Summary It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm³ can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50°C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.     

Variation of lung volume after fixation when measured by immersion or Cavalieri method

Organ volume is a critical parameter in morphometric analysis. The special problems of the lung as a nonsolid organ are overcome by tracheal instillation of fixatives at a constant airway pressure (P(aw)). Lung volume can change significantly after fixation as P(aw) change. To determine the variation of lung volume after fixation, we measured the volume of intact fixed lungs by serial immersion in saline (V(imm)) at selected time points, compared with measurements obtained by point counting [Cavalieri Principle (V(cav))] after tissue sectioning to release P(aw). V(imm) was systematically higher than V(cav) by 25% in dog lungs and 13% in guinea pig lungs (P = 0.0003 between species). This size-dependent variability reflects residual elastic recoil, refolding and/or crumpling of alveolar septa after fixation. V(imm) remained 14% higher than V(cav) in dog lungs even after pressure release. V(cav)/V(imm) was systematically lower in the upper than the lower strata of the same lung. We conclude that V(cav) measured on lung slices after relaxation of P(aw) more precisely represents the state of the tissue to be used for subsequent morphometric analysis, particularly for large lungs.