Application of alternative fixatives to formalin in diagnostic pathology

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

Summary A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about onethird those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried metarial fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.     

STUDIES ON FORMALIN FIXATION FOR ELECTRON MICROSCOPY AND CYTOCHEMICAL STAINING PURPOSES

A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0–2°C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Electron-microscope studies of Drosophila salivary-gland chromosomes

Summary The submicroscopic structure of the chromosomes of the larval salivary gland cells inDrosophila melanogaster has been studied by means of electron microscopy of thin sections.The three fixatives used were buffered 1% osmium tetroxide, unbuffered Zenker's solution, and buffered formaldehyde in concentrations of 0·1% and 1·0%. It was found that formalin preserves the fine structure of the chromosomes better than the other fixatives.Nucleoprotein is present in the chromosome in the form of granules 200 Å to 300 Å in diameter. The granules are connected together by strands of protein approximately 100 Å in diameter. The length of the strands between successive granules in the banded areas is 100 Å to 300 Å and in the interband regions it is 700 Å to 900 Å.Heterochromatin differs from euchromatin in the absence or reduction in the amount of strand material.The nucleolus is composed of an aggregate of granules 300 Å to 600 Å in diameter contained in an amorphous matrix. Within the nucleolus are a series of canals containing material similar in appearance to that of the euchromatic band material.Submicroscopic granules are also present in the karyoplasm and in the cytoplasm surrounding the nuclear membrane. The cytoplasmic granules are more densely distributed near the nuclear membrane.A model for chromosome structure is proposed.The Applied Fisheries Laboratory is operated by the University of Washington under Contract No. AT(45-1)540 with the United States Atomic Energy Commission.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

Summary We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I,-III and-IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and-III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraftin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Concentrated Formalin Versus A 10% Solution as a Fixative Preceding Silver Staining

The effect of concentrated and 10% formalin as fixatives for the silver staining of axons in the central nervous system in a series of mammals has been compared. It is felt that concentrated formalin constitutes a better fixative for this purpose in several respects: it has a greater speed of penetration without causing additional distortion or artifacts, the staining time can be reduced somewhat and the normal configuration of the axons is better preserved particularly in the largest ones. Satisfactory results were obtained following 51/2hours fixation in concentrated formalin. Further, concentrated formalin is at least equal to a 10% solution in the ease in which paraffin and frozen blocks can be sectioned, in the clearness of the background of stained sections and in the consistency of results.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and -III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraffin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Effects of Various Fixing and Storage Fluids on Starch in Sapwood

Wood material that is to be used for observation of starch content may be stored many months in several plant material fixatives, formalin-acetic-alcohol, formalin-propionic-alcohol, tertiary butyl alcohol, ethyl alcohol, and 5 and 10% aqueous formalin without any apparent deleterious effect on the starch granules Storage of wood in aqueous formalin solutions of 15% and higher concentrations caused starch granules to break down and disperse forming a coagulated mass, in which it was impossible to see the individual grains.     

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.     

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

Summary 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 as 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.     

Non-specific esterase activity in bovine acrosomes

Synopsis Fixed and unfixed smears of bovine spermatozoa were stained for esterase activity at pH 6.1–6.2 using 1-naphthyl acetate as substrate and hexazonium pararosaniline as coupler. Best results were obtained with unfixed smears prepared from suspensions of spermatozoa washed free of seminal plasma. The most intense staining was obtained when smears were incubated at 4°C for 40 hr with replacement of the incubating medium every 12 hr. No staining was detected in spermatozoa when smears were incubated in medium lacking substrate or when heat-inactivated smears were incubated in complete medium. Enzyme activity was restricted to the acrosomal cap; no final reaction product was detected in or on the post-nuclear cap, the mid-piece or the remainder of the flagellum. Enzyme activity was distributed quite uniformly throughout the acrosome with no indication of the existence of localized regions with high activity. The use of fixatives, especially those containing formalin, is not recommended for esterase studies of bovine spermatozoa.     

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.     

Immunofluorescence Microscopy of Cyanurated Tissues

Test tissues consisted of: (1) popliteal lymph nodes of rabbits, removed 6 hr after injection of hind footpads with 0.2 ml of 125 mg/ml solution of 5× crystallized chicken ovalbumin, and (2) lungs from guinea pigs, passively sensitized with rabbit antiovalbumin serum, then anaphylactically shocked by intracardial injection of a 1% chicken ovalbumin solution. Similar control tissues from normal rabbits, and lungs of passively sensitized guinea pigs, but shocked with histamine instead of ovalbumin, were included. Pieces of fresh tissue not exceeding 2 mm³ were fixed as follows: (1) Cyanuration—lymph nodes, 1 hr; lung, 0.5 hr; both at 23-27 C—in anhydrous methanol containing 0.5% w/v cyanuric chloride and 1% v/v N, N-diethylaminoethanol. (2) Control fixatives—all specimens 18-24 hr at 4—6 C—absolute methanol; 95% ethanol; neutral buffered 10% formalin; and an FAA mixture (formalin, conc., 6; glacial acetic acid, 2; 30% ethanol, 92). Freeze-dried material was either left unfixed (a control) or fixed in xylene or toluene containing 0.5% w/'v cyanuric chloride and 1% v/v N, N-diisopropylaminoethanol; time and temperature as for fresh tissues. All tissues were routinely dehydrated, cleared, and vacuum embedded in an ester wax, diethylene glycol distearate, or in paraffin at 52 C. Sections 2-4 μ thick were attached to gelatin-coated slides, the wax removed in petroleum ether, and stained 20 min at 23-27 C in a 0.10% solution of fluorescein isothiocyanate-conjugated rabbit antiovalbumin globulin, washed in phosphate buffered saline 10 min, dehydrated, cleared and covered in a nonfluorescent medium. With ultraviolet illumination, brightly immunofluorescent, anti-genically specific staining was obtained in cyanurated fresh and freeze-dried lymph node and lung tissues. In contrast, specific staining was diminished or absent in comparable tissues reacted in the control fixatives.     

A Glycine-Hcl-Formalin Fixative for Improved Staining of Neuroglia

The pH of fixatives as well as the components of the fixative mixture exert a displacing action on the isoelectric point of tissue proteins, which influences the intensity of staining. Studies of these effects showed that ammonium nitrate, sulfate or chloride could be substituted successfully for ammonium bromide in Cajal's formalin-ammonium bromide fixative. However, the best results from staining with Rio Hortega's silver carbonate and with Cajal's gold-sublimate methods were obtained after fixation in a mixture consisting of: glycine, 1.05 gm; 12V HCl, 14.8 ml; concentrated formalin, 15 ml and distilled water to make 100 ml.     

Effects of fixation, dehydration and staining on dimensions of myxosporidan

The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores.