Determination of optimal rehydration,fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde.

Liver tissue from miniature pig fetuses was immersion-fixed in fixative mixtures with various concentrations of formaldehyde and glutaraldehyde. The preservation quality of hepatocytes was evaluated ultrastructurally in a peripheral zone (30--130 micron below the surface) and a central zone (500 micron below the surface). In the peripheral zone the best preservation was obtained with a fixative mixture containing 2% formaldehyde and 2% glutaraldehyde and in the central zone with a fixative mixture containing 8% formaldehyde and 8% glutaraldehyde. It is concluded that a better utilization of fairly large tissue blocks for ultrastructural investigation can be obtained by division of the block and subsequent fixation in fixatives containing various concentrations of formaldehyde and glutaraldehyde.     

Preservation of glycogen in decalcified hard tissues

Summary Various fixatives and fixation procedures were tested to evaluate their effects on the preservation of glycogen in sections of decalcified hard tissues. Lower jaws from 1-day-old rats were chosen for the observations. An aqueous solution of glutaraldehyde showed poor preservation of glycogen in the tissues even when employed in the perfusion procedure. Freeze-drying and formaldehyde vapour fixation preserved it much better, but glycogen was still lost to some extent. Freeze-substitution with acetone and various alcoholic fixatives gave a poor result, unless the tissues were fixed with cyanuric chloride. Cyanuric chloride in methanol containing N-methyl morphorine was the best fixative for the preservation of glycogen in the sections. A combination of freeze-substitution with the cyanuric chloride solution, decalcification with the Jenkins's fluid, and subsequent double-embedding in celloidin and paraffin was recommendable for an excellent glycogen preservation.     

Histomorphometric comparison after fixation with formaldehyde or glyoxal

Abstract

Formaldehyde has long been the fixative of choice for histological examination of tissue. The use of alternatives to formaldehyde has grown, however, owing to the serious hazards associated with its use. Companies have striven to maintain the morphological characteristics of formaldehyde-fixed tissue when developing alternatives. Glyoxal-based fixatives now are among the most popular formaldehyde alternatives. Although there are many studies that compare staining quality and immunoreactivity, there have been no studies that quantify possible structural differences. Histomorphometric analysis commonly is used to evaluate diseased tissue. We compared fixation with formaldehyde and glyoxal with regard to the histomorphological properties of plantar foot tissue using a combination of stereological methods and quantitative morphology. We measured skin thickness, interdigitation index, elastic septa thickness, and adipocyte area and diameter. No significant differences were observed between formaldehyde and glyoxal fixation for any feature measured. The glyoxal-based fixative used therefore is a suitable fixative for structural evaluation of plantar soft tissue. Measurements obtained from the glyoxal-fixed tissue can be combined with data obtained from formalin-fixed for analysis.     

Formaldehyde-amine fixatives for immunocytochemistry of cultured Xenopus myocytes

Although formaldehyde is commonly used in immunocytochemical studies, this fixative can cause distortions in cell structure. We tested the possibility that adducts of formaldehyde and primary amines could be used as improved fixatives for immunolabeling studies of cultured cells. A variety of primary amines were reacted with formaldehyde and applied to cultured Xenopus muscle cells, after which the cultures were labeled for immunofluorescence. Amine-formaldehyde fixatives improved structural preservation of the myocytes as compared with formaldehyde alone. The extent of improvement depended on the amine tested; the best results were obtained using cyclohexylamine. Immunofluorescence localization of a variety of antigens was better in myocytes fixed with cyclohexylamine-formaldehyde than in cells fixed with formaldehyde alone. In addition, the fixative provided good ultrastructural preservation of cytoskeletal structures and permitted immunogold labeling for alpha-actinin by use of pre-embedding labeling techniques.     

Optimizing specimen processing for ancient soft tissue specimens

Abstract

Despite many reports concerning processing of ancient soft tissues, scant attention has been paid to optimizing procedures for processing soft tissues that have been altered by taphonomic processes. To determine the best procedures, we investigated the rehydration solution, time of exposure to the solutions, fixative solution and exposure to heat. Processes were evaluated based on the minimum section thickness, degree of tissue fragmentation, definition of tissue architecture and penetration of stains. We found that in desiccated samples, tissue architecture was optimized by using Ruffer's solution for rehydration and Schaffer's solution as fixative, because these tissues require water restoration within the tissues due to their compacted character. Heating enhanced penetration of dyes in these specimens, which improved diagnosis. Saponified tissues that had suffered extensive decomposition were more labile and required slow water uptake. The best histological sections were obtained using Sandison's solution followed by fixation with formaldehyde and avoiding heat. To obtain the best results with paleohistological specimens, the procedure must be determined by the condition of the sample and by accounting for the nature of its damage.     

Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.     

Influence of prefixation and fixation times on cellular preservation and epithelial cellularity in filter imprints

Cells from malignant and nonmalignant lesions of the breast were suspended in three different fixatives or in a balanced electrolyte solution (Hank's), stored for varying periods of time, collected on Millipore filters and then imprinted on to clean microslides in order to evaluate the influence of prefixation and fixation time on epithelial cellularity and cellular preservation. The use of a methanol-acetic acid fixative (Esposti's fixative) or 50% isopropanol resulted in good preservation whereas cells prefixed in formaldehyde or 100% isopropanol were poorly preserved. Cells that had not been prefixed (suspended in Hank's solution) showed fair preservation. Eighty-eight percent of the imprints prepared from suspensions of Esposti's fixative were highly cellular, which was significantly better than with Hank's solution (68%), 50% isopropanol (66%), 100% isopropanol (56%) and formaldehyde (33%). The cellularity of the formaldehyde-prefixed imprints differed significantly from the others. There was no influence of storage time on either cellular preservation or epithelial cellularity for any of the investigated solutions.     

Liquid Detergent in Aqueous Fixing Fluids, to Promote Rapid Wetting and Sinking of Plant Specimens

Plant tissue immersion for fixation is facilitated by the addition of a drop or two of liquid detergent to each 10 ml of fixing solution. Six fixatives: FAA, chrome-acetic, Craf I, III, V and Farmer's were tested with 1-7% additives of a liquid detergent (Joy was used). All were found to facilitate wetting and sinking of the specimens in the aqueous fixatives. Farmer's fixative (alcohol-acetic 3:1), however, did not require detergent. The detergent did not harm the tissue nor affect staining.     

Temporal bone morphology after systemic arterial perfusion or intralabyrinthine in situ immersion—I. Hair cells of the vestibular organs and the cochlea

The morphological preservation of hair cells of the inner ear was analysed following fixation with intra-arterial perfusion or local intralabyrinthine in situ immersion using six different fixatives of glutaraldehyde (441–876mosm/kg), three fixatives with combinations of glutaraldehyde/paraformaldehyde (917–1196mosm/kg) and 1% osmic tetroxide (346mosm/kg). The influence of added macromolecules such as dextran to the buffer solution was investigated with regard to fixation technique and type of fixative.The best results were obtained when using a direct (local) immersion of the labyrinth with 1% osmic tetroxide. Vascular perfusion is not recommended, independent of type of fixative used. Cochlear outer hair cells and vestibular hair cells type I are more vulnerable to alterations of osmolality and fixation technique than are inner hair cells and hair cells type II.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

The effect of formaldehyde containing fixatives on ribonuclease activity

Summary 1. The inactivation of crystalline ribonuclease by formaldehyde and formaldehyde containing fixatives (Serra's solution) is demonstrated.2. The rate of inactivation is shown to be dependent uponph, formaldehyde concentration, and time of action of the fixative.3. The effect of formaldehyde containing fixatives on the RNase activity in sections from fixed tissues is discussed, and the inactivation of that enzyme system in rat pancreas is demonstrated.With 2 Figures in the Text     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Effects of Fixation on Cell Volume of Marine Planktonic Protozoa

The effects of fixation on the cell volume of marine heterotrophic nanoflagellates and planktonic ciliates were investigated. Decreases in cell volume depended on the combination of the protozoan taxa and the particular fixative. For a particular fixative and protozoan species, degree of shrinkage was independent of physiological state. The volume of fixed cells was found to be approximately 20 to 55% lower than the cell volume of live organisms. For the heterotrophic microflagellates, the fixatives ranked, in order of decreasing effect on cell volume, as glutaraldehyde, formaldehyde, acid Lugol's solution, and modified van der Veer solution. With oligotrichous ciliates and a tintinnid ciliate, formaldehyde caused less shrinkage than glutaraldehyde or acid Lugol's solution. With the aldehyde fixatives, the microflagellates were found to shrink more than the ciliates. Differential effects of fixation on cell volumes may result in an underestimation of the biomass of certain protozoan taxa in natural samples.     

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections

Summary The AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoy's fluid, Bouin's fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Formaldehyde fixation and microwave irradiation

Summary Formaldehyde is the most commonly used fixative in pathology laboratories. However, due to time pressures, this fixative is often not optimally exploited. the majority of biopsies are only partly fixed when histoprocessing is started, with adverse effects. This paper reports how formaldehyde fixation is improved, by using 1.5 min of microwave irradiation of tissue previously soaked for four hours in the fixation solution. It is argued that this beneficial effect of microwave irradiation can be attributed to the acceleration of the reaction of formaldehyde to the tissue. Formation of free formaldehyde, by the dehydration of methylene glycol present in the tissue when the irradiation starts, is also enhanced. Five different formaldehyde-containing fixatives were evaluated, using five different working protocols. Spleen was taken as a suitable tissue for these tests. The technique described leads to uniform microscopical results. It is a simple method and is suitable for use in routine laboratories.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

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.