Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells

KleinJan et al, (Allergy 1996;51:614-20) reported that Carnoy's fixative reduced the number of chymase-positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoy's fixative reduces the number of chymase-positive cells from cord-blood-derived human cultured mast cells when compared with other types of fixatives. Human mast cells were obtained by culturing cord-blood-derived CD34-positive cells in the presence of stem cell factor and interleukin-6. Staining procedures of the cells in fixation with Carnoy's fixative and with other fixatives gave no differences among the number of tryptase-positive cells, whereas fixation with Carnoy's fixative for 15 min gave a significant decrease i n the number of chymase-positive cells compared with acetone for 10 min. The number of chymase-positive cells decreased in a time-dependent manner under fixation with Carnoy's fixative, indicating that Carnoy's fixative had a negative effect on the number of chymase-positive cells from cord-blood derived human cultured mast cells.     

Lung deformation and macrophage displacement in smoke-exposed and normal mice (Mus musculus) following different fixation procedures

Summary Lung deformation (shrinkage or inflation) and displacement of pulmonary parenchymal macrophages were evaluated after immersion fixation, intratracheal instillation of fixative and lung lavage followed by intratracheal fixative instillation in cigarette smoke-exposed, sham-treated and control pallid male mice. Lung volume displacement and lung section and alveolar area analysis revealed that degree of deformation was uniform in lungs from all treatment groups fixed by immersion but not by instillation of fixative and fixative instillation following lavage. In situ pulmonary parenchymal macrophage number per lung section area of fixative-instilled lungs and lavaged lungs followed by fixative instillation was significantly greater than in those following immersion fixation in all corresponding treatment groups. A paucity of macrophages was noted in airways of fixative-instilled and lavaged followed by instillation of fixative lungs. Pulmonary macrophages were uniformly distributed throughout lung parenchyma following immersion fixation, while in fixative-instilled and lavaged prior to instillation of fixative lungs these cells tended to be concentrated in alveoli near terminal bronchioles. Lavage procedures removed an unknown portion of lung macrophages and appeared to ineffectively sample the pulmonary parenchymal macrophage population. Intratracheal instillation of fixative with or without prior lavage apparently alters the distribution of pulmonary macrophages by displacing airway phagocytes into the alveoli. Data reported suggest that fractional estimates of in situ lung parenchymal macrophage population can be obtained by counting the number of these cells per area of tissue from lungs fixed by immersion.     

Molecular fixative enables expression microarray analysis of microdissected clinical cervical specimens

Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50 ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis.     

Pol André Bouin, MD (1870-1962). Bouin's fixative and other contributions to medicine.

Pol André Bouin was a distinguished French scientist who gained international reputation at the turn of the century for his work in histology and reproductive endocrinology. His name has been retained eponymously in the fixative he described in 1897, today known as "Bouin's fixative." The fixative that bears his name is only one of many contributions he made to academic medicine.     

Rapid method for fetal brain fixation.

A quicker alternative to the standard removal and fixation of brain tissue was sought. Whole fetal brains were fixed in situ using a mercuric based fixative. The subarachnoid space was perfused overnight with Heidenhain's Susa fixative. The following day the brains were removed from the cranium in the standard manner. After storage for three days in Susa's fixative the brain was sliced and processed, with excellent preservation of gross and microscopic architecture. The cost is only marginally higher than that of commercial formalin.     

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.     

Liver Mitochondria in Health and Disease

Abstract

Samples of formaldehyde fixative solutions were taken from a random selection of 100 tissues submitted to a large tissue repository. The solutions were tested for formaldehyde concentration, pH, osmolality, content of organic solvents and buffering capacity. There were considerable variations in the fixative samples with respect to all variables tested. While all tissue repository specimens examined were adequate for histopathology, this adequacy was presumably due to extended formaldehyde immersion, a luxury not often enjoyed in a clinical setting. Simple analytical procedures within the reach of many clinical laboratories are described for testing fixative solutions.     

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.     

A random arrangement of albumin-containing hepatocytes seen with histo-immunologic methods. II. Conditions that produce the artifact

The cellular immunolocalization of albumin in rat liver has been studied as a function of various physiological and physical conditions. Our observations show that the prime requisite for accurate immunolocalization of albumin and other hepatic-based proteins is the complete removal of blood and especially plasma from sinusoids and the perisinusoidal space of Disse prior to fixation. Fixation of blood-filled liver specimens results in the antifactual entrance of plasma constituents into hepatocytes. When the fixative used is formaldehyde, the artifactual uptake occurs primarily into hepatocytes that have a high glycogen content. Fixation of blood-filled liver with acetic acid-ethanol causes a massive influx of plasma into all hepatocytes. On the contrary, with blood-free liver, varying the type of fixative consistently demonstrates that all hepatocytes normally contain albumin, transferrin, and fibrinogen simultaneously. Increasing the time between cessation of blood flow and outright fixation by either withholding the fixative or by impeding its diffusion through the specimen causes a progressive loss of antigenicity of albumin. The same result ensues when specimens remain in contact with the fixative for an extended time.     

The Effect of Various Fixatives and Trypsin Digestion Upon the Staining of Routine Paraffin-Embedded Sections by the Peroxidase-Antiperoxidase and Immunofluorescent Technique

Abstract

The use of immunohistochemical techniques in biological and diagnostic pathology laboratories, and the theory of the unlabelled peroxidase-antiperioxidase technique, is briefly reviewed. The results of experiments using a variety of routine fixatives, with pretreatment of the sections with a trypsin solution, indicated that while Bouin's fixative alone was superior to formalin fixation, the pretreatment of sections with a trypsin solution gave markedly improved results with either fixative — the results with buffered formalin then being almost equal to those after Bouin's fixative. This methodology allows both prospective and retrospective immunohistochemical studies to be made on routinely fixed and processed paraplast-embedded tissues using the light microscope.     

Color preservation of gross specimens for teaching and medical illustration

We routinely employ cold Jores' fixative to improve color preservation of gross autopsy specimens for conferences and teaching and to facilitate development of a photographic teaching collection of gross pathological specimens. The fixed specimens also permit light microscopic examination. Additionally, we have found that color restoration of previously fixed gross specimens is possible with McCormick's fixative.     

An antibody panel for immunohistochemical analysis of the retina in Davidson's-fixed,paraffin-embedded eyes of rats

Unlike most other tissues, the optimal fixative for preserving eye morphology is considered to be Davidson's fixative or modified Davidson's rather than formalin. However, the methodology for antibodies to be used in tissues fixed this way is not normally outlined in current antibody datasheets. Additionally, where eyes have been stored in Davidson's fixative, the efficacy of retrospective analysis of eye morphology by immunohistochemistry is largely unknown. The aim of this study was to compare a panel of six antibodies in both Davidson's-fixed and formalin-fixed pigmented and non-pigmented rat eyes, in order to provide optimal methods for future retinal immunohistochemical evaluation with image analysis. The antibodies evaluated were raised against rhodopsin, synaptophysin, glutamine synthetase, glial fibrillary acidic protein (GFAP), cleaved caspase-3 and phospho-histone H3 (PH3). Overall, the staining quality of these antibodies was found to be optimal in Davidson's compared to formalin-fixed tissues after a time period of up to 4 days in fixative. The methods outlined thus provide a platform for future detailed analysis of retinal pathology in Davidson's-fixed eyes.     

Fixation of the macula densa with fixatives of different osmolarities in normal and diabetic rats.

The influence of different fixative osmolarities on the ultrastructure of the macula densa region has been studied in normal and in streptozotocin diabetic rats after 50 days' duration of diabetes. In vivo perfusion fixation of the kidneys was performed retrogradely through the aorta in five groups of controls and diabetic animals with a 1% glutaraldehyde fixative in a Tyrode solution. Between groups, the osmolarity of the fixative solution varied from 216 to 476 mosm/l. This was accomplished by variation of the NaCl concentration of the fixative vehicle. The tissue was embedded in Epon and midcortical tissue was prepared for electron microscopy. The volume density of the lateral intercellular spaces between the macula densa cells was measured applying a morphometric technique. The results confirmed that the lateral intercellular spaces are narrowed in diabetes and also showed that for all fixative osmolarities the difference is persistent between diabetic and non-diabetic animals, except at very low osmolarities. The abnormal macula densa ultrastructure has been suggested to be connected with the functional abnormalities in diabetes, i.e. the resetting of the tubuloglomerular feedback and subsequent increases in GFR. It is therefore of interest to further understand the mechanisms underlying the narrowing of the lateral intercellular spaces in diabetes. The present study has shown that the size difference in lateral intercellular spaces between diabetic and normal animals is reproducible over a wide range of fixative osmolarities, further indicating that it is an in vivo phenomenon. The diminution of the LIS in diabetes could perhaps indicate a decrease in signal function, previously reported at the MD level in diabetic animals, contributing to the increased GFR in diabetes.     

NSH in Action

Abstract

An S-shaped hook fashioned from wire was hung from a strip of polystyrene to suspend brains in fixative. The material was obtained from packaging and wire already found in the laboratory. This simple and economical method allowed total immersion of the brain and free access to fixative. Fixation was completed without damage or distortion of tissue.     

Demonstration of amyloid protein AA in old museum specimens

Three amyloid-infiltrated organs, which had been stored in fixative for 65 to 83 years, were obtained from a collection of pathologic tissues. All the tissues originated from persons with pulmonary tuberculosis. Amyloid fibril protein AA, typical of secondary systemic amyloidosis, was demonstrated in all the tissues by the peroxidase-antiperoxidase method. Protein AA was also extracted and purified from the tissues in spite of the long period in fixative.     

Antimicrobial activity of UMFix tissue fixative

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.     

Fine structure of the rat renal papilla

The fine structure of the rat renal papilla was studied after fixation with various fixative solutions and by using three modes of fixative application: immersion, vascular perfusion and injection of fixative into the renal pelvis. The morphology of the following was described: thin limbs of Henle's loop, collecting ducts, capillaries, interstitial cells, and interstitium. Several previously undescribed features were seen. Two types of capillaries were identified on the basis of their fine structure. Regions identified as limbs of Henle had at least two types of mural structure differing in degree of cellular interdigitation. In addition, numerous projections were seen extending from the basal cytoplasm of capillary endothelium and collecting duct epithelium through the basement membrane into the interstitium.     

Lymph vessels of the heart. A comparative study of various techniques for light and electron microscopy

This investigation was carried out in order to find out which was the best technique, among those reported in the literature, to recognize the small lymph vessels by light microscopy, while at the same time allowing a good ultrastructural preservation of tissues. The study was performed with rabbit hearts according to the following methods: (1) injection of India ink into the wall of the heart followed by perfusion with aldehyde fixative; (2) injection of India ink into the wall of the heart followed by immersion in aldehyde fixative; (3) treatment of the animals with histamine dihydrochloride by e.v. route and aldehyde (4) fixation by immersion; (5) fixation by immersion in osmium tetroxide fixative; fixation by immersion in aldehyde fixative; (6) fixation by perfusion with the same mixture as in (5). In cases (1), (2), (3), (5), and (6), the tissue specimens were postfixed in osmium tetroxide according to conventional methods. The validity of the different methods was checked by examining semithin sections of varying thickness at the light microscope and ultrathin sections at the transmission electron microscope. A comparative examination of the preparation showed that the method allowing the promptest recognition of the small lymph vessels, together with the best ultrastructural preservation and a good reproducibility of results was perfusion of the coronary circle.     

A novel fixed paraffin-embedded tissue processing for proteomic analysis

Human tissues are an important biological material for the discovery of biomarkers and identification of novel therapeutic targets. Formalin fixed and paraffin embedded (FFPE) tissue represents the most abundant supply of archival material for clinical and molecular analyses. Although FFPE preserves the cellular and architectural morphologic details in tissue sections, formalin facilitates the formation of protein-protein crosslinks rendering FFPE tissues refractory to many protein studies. The aim of this study was to assess the feasibility of proteomic investigations of a new non-toxic fixative using a comprehensive panel of proteomic methods. Tissues were processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Similar protein patterns were observed between our tissue fixative protocol and frozen tissues using mono and bidimensional electrophoresis and protein identification by mass spectrometry was not affected. Several proteins were successfully detected using western blot and immunohistochemistry showed comparable results between both tissue storage methods. We demonstrate that our new fixative protocol represents an easy-to-use alternative to FFPE compatible with both current diagnostic pathology practice and tissue proteomic investigations.     

Fixation temperature affects DNA integrity in the testis as measured by the TUNEL assay

Mature rat testes and liver were fixed with Bouin's fluid (BF) or modified Davidson's fixative (mDF) at room temperature (23 °C) or 4 °C, and DNA integrity was examined by the TUNEL assay. When testes were fixed in BF, TUNEL-stained cells were more prevalent than when fixation occurred in mDF. Independent of fixative, TUNEL-staining was higher when testes were fixed at room temperature relative to 4 °C. Significant effects were present for fixative and temperature of fixation, but not their interaction. Relative to BF, mDF also provided for lower TUNEL-staining in liver, but staining was not affected by fixation temperature. Since the TUNEL assay depends on the detection of fragmented DNA strands, harsh fixatives that induce breaks in the DNA can introduce substantial artifacts. Such potential artifacts are especially prevalent in a tissue such as testes with its ongoing division and differentiation activities. Therefore, the current findings lead the authors to conclude that fixation of mature testes in mDF at 4 °C minimizes generation of false TUNEL-positive cells.