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.     

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 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.     

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 effects of various fixatives on subsequent lectin binding to tissue sections

Summary The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin—saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate—paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin—saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.     

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 immuno-fluorescent 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 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 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

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.     

Variability of laminin immunoreactivity in human autopsy brain

Summary Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

Alteration of immunoglobulin-bearing lymphoma cells by fixation.

Four large cell lymphomas known to be monoclonal B-cell proliferations were studied with immunofluorescent and immunohistochemical methods for the detection of kappa- and lambda-light chains. Frozen sections of lymphoma tissues as well as formalin and B-5-fixed tissues embedded in paraffin were studied. Both immunofluorescent and immunohistochemical methods gave similar results on frozen sections; however, a number of discrepancies were noted between the results obtained on fixed tissues and those obtained on frozen tissues. In an effort to identify a fixative which did not alter immunoglobulin (Ig), mouse lymph nodes were fixed in different fixatives before Ig detection; but all of the fixatives tested destroyed the Ig present on normal cortical B lymphocytes. Immunoglobulin-bearing normal and neoplastic lymphocytes are better detected on frozen sections than on paraffin sections after routine fixation.     

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.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique

The influence of testicular hyaluronidase treatment on the immunohistochemical localization of fibronectin in different tissues (human articular cartilage, large intestine, synovial membrane and experimental granulation tissue) as well on frozen as on formaldehyde fixed, paraffin embedded tissue, has been studied using the indirect immunoperoxidase technique. Pretreatment with hyaluronidase is essential in demonstrating fibronectin in frozen sections of human articular cartilage. In the other tissues examined treatment with hyaluronidase was not essential, but gave a more optimal staining quality. The effect of hyaluronidase treatment was to some extent dependent on the duration of treatment. In formaldehyde fixed, paraffin processed tissue the improvement with hyaluronidase treatment was only seen when the hyaluronidase followed pepsin digestion of the deparaffinized tissue sections.     

The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique

Summary The influence of testicular hyaluronidase treatment on the immunohistochemical localization of fibronectin in different tissues (human articular cartilage, large intestine, synovial membrane and experimental granulation tissue) as well on frozen as on formaldehyde fixed, paraffin embedded tissue, has been studied using the indirect immunoperoxidase technique. Pretreatment with hyaluronidase is essential in demonstrating fibronectin in frozen sections of human articular cartilage. In the other tissues examined treatment with hyaluronidase was not essential, but gave a more optimal staining quality. The effect of hyaluronidase treatment was to some extent dependent on the duration of treatment. In formaldehyde fixed, paraffin processed tissue the improvement with hyaluronidase treatment was only seen when the hyaluronidase followed pepsin digestion of the deparaffinized tissue sections.     

Tissue fixation with phenol-formaldehyde for routine histopathology

Summary The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol—formaldehyde (pH7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol—formaldehyde buffered to pH7.0 and pH5.5 was carried out at an elevated temperature (40°C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol—formaldehyde (pH7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol—formaldehyde (pH7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol—formaldehyde (pH7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol—formaldehyde (pH7.0) and neutral buffered 4% formaldehyde showed similar volume changes.     

Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.     

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.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

Comparison of the effects of two fixatives for immunolocalization of testosterone in the testes of the cynomolgus monkey, mouse and rat.

We compared the effect of two fixatives, Bouin's fixative and neutralized buffered 4% formaldehyde (10% formalin), for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats. In the samples fixed with Bouin's fixative, immunoreactive testosterone was detected as intense deposits in the cytoplasm of Leydig cells of monkeys and mice. Immunoreactive testosterone was detected not only in Leydig cells of rats but also moderately shown within tubules. Immunoreactive testosterone could not be detected in the testes of monkeys, mice or rats fixed with neutralized buffered formalin because of the poor morphology caused by the fixative. It is concluded that Bouin's fixative is a suitable fixative for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.