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.     

Embedding and Fixation Techniques for Immunohistochemical Staining with Anti-DNA Polymerase α and Ki-67 Monoclonal Antibodies to Analyze the Proliferative Potential of Tumors

Anti-DNA polymerase a and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.     

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 prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Influence of season on the incidence of DNA hypodiploidy in urinary cytology

OBJECTIVE: This study was conducted to determine if an incidence of hypodiploidy in urinary specimens is related to seasonal temperature changes. MATERIALS AND METHODS: DNA ploidy was evaluated on 10,846 urinary specimens fixed in buffered alcohol (MOPSO/NaCl + ETOH) and received over a one year period from numerous sites throughout the United States. The percentage of hypodiploid (DNA index < 0.8) cases was evaluated in each month. As a control, DNA ploidy results from 3, 755 prostate biopsies, fixed in 10% neutral buffered formalin, received during the winter and summer months of the same year, were evaluated. RESULTS: The average percentage of hypodiploidy in cytologic specimens during the summer months was 19.6% compared to 5. 4% in the winter and early spring months (range: 20.6-4.8%). The average percentage of hypodiploid cells in histologic specimens was 0.8% for both the summer and winter months (range: 1.73-0.36%). CONCLUSIONS: The rate of hypodiploidy in urinary cytology seems to be temperature related. The hypodiploidy rate of histologic specimens fixed in formalin shows no fluctuation with the seasons.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01-0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

Increase in Feulgen staining intensity by pre-treatment with different reagents.

In the experiments sections of rat tissues fixed in 10 per cent buffered neutral formalin for 3 hour and treated for 15 minutes with different chemical reagents such as pyridine, tributylamine, urea, tris sodium nitrite and sodium hydroxide were subjected to hydrolysis in 6 N HCl at 25 degrees C for 20 minutes and stained by the UV Feulgen technique. The results reveal a far more intense staining of the nuclei in tissues treated with any of the chemicals mentioned than in untreated controls. The possible role of these chemicals in enhancing the staining intensity is discussed.     

The effect of prefixation on the diameter of chromosome fibers isolated by the Langmuir trough-critical point method

The effect of prefixation on the diameter of chromosome fibers isolated by the Langmuir trough-critical point method has been investigated in several species of plants and animals. In barley, fibers isolated from endosperm without prefixation have an average diameter of between 240 and 250 A, and are similar in dimensions and structure to the chromosome fibers isolated from animals by this method. Chromosome fibers from other tissues of the same plant are smaller in diameter when isolated without prefixation, approximating 200 A. After prefixation in 2% buffered formalin, isolated fibers from the three barley tissues studied are reduced in diameter, to approximately 120-130 A for endosperm and leaflet and to 140 A for root tip. Chromosome fibers isolated from newt erythrocytes also show a significantly reduced diameter after formalin prefixation, to approximately 120 A.     

Rapid microwave fixation--a comparative morphometric study

Summary Tissue blocks taken from healthy human lung tisues, from primary bronchial carcinoma and from mediastinal and hilar lymph nodes were placed in the following solutions. Tris buffer; buffered formalin (0.5%, 1%, 7%); 0.1 mol NaCl; distilled water; DMSO (1%, 10%, 20%); acetone (10%); methanol (50%, 80%, 100%); glutaraldehyde (2.5%), and fixed by use of a commercial microwave oven.Tissue blocks obtained from the same surgical specimens were fixed in 7% buffered formalin for 24 h for comparison. Conventional and microwave-fixed tissue was embedded in paraffin, and stained with haematoxylin and eosin. Fifteen specimens of each group and each solution were examined by light microscopy. Minimum diameter and area of 100 nuclei of each specimen were measured interactively.Histomorphological sections fixed with Tris buffer in a microwave oven revealed best morphological results showing more contrast in chromatin distribution of nuclei and opening of interstitial lung capillaries in comparison to conventional formalin-fixed specimens. No statistically significant differences in area and minimum diameter of nuclei between the different groups were found. Microwave fixation using Tris buffer is a time-saving fixation method at least comparable to conventional formalin fixation. It is not accompanied by hazards to the environment that are unavoidable by formalin fixation.     

Museum fish specimens and molecular taxonomy: A comparative study on DNA extraction protocols and preservation techniques

Museum fish specimens are invaluable resources for genetic studies, but extraction of high quality DNA is often problematic. In this study, hairtail fishes of the genera Trichiurus and Lepturacanthus (family: Trichiuridae) representing a wide range of preservation histories and three different methods of preservation were analyzed for mitochondrial DNA (mtDNA) extraction, amplification and sequencing of marker genes. A total of six protocols, including a commercially available kit, were compared in this study. Amplification of conserved genes such as16S rRNA and 12S rRNA were done using polymerase chain reaction with sequence analyses using automated capillary sequencing techniques. The results show that mtDNA extraction, amplification and sequencing of conserved genes could be obtained successfully from frozen (?20°C) preserved specimens (1–5 years) and also from ethanol (95%) fixed specimens (2–5 years) but not from any of the formalin (10%) fixed specimens (3–4 years). However, specimens that have been fixed for only 7 days in buffered formalin (10% formalin with phosphate buffer containing 173 mm salt) and ethanol (95%) could yield successful mtDNA extraction, amplification and sequence information of both 16S rRNA and 12S rRNA.     

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 Antigen Retrieval on Cells Fixed in 10% Neutral Buffered Formalin Followed by Transfer to 70% Ethanol

Fixation in 10% neutral buffered formalin prior to transfer to 70% ethanol for one week has been shown to adequately preserve immunorecognition of PCNA, cytokeratins AE1/AE3 and EGFr. This study investigated whether 12 hrs fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks would similarly preserve immunorecognition to an extent where antigen retrieval (AR) used to reverse the masking effects of fixation on some antigens would not be necessary. Two cell lines, DU145 and SKOV3 were grown on coverslips and fixed either for 684 hrs in 10% NBF or for 12 hrs in 10% NBF which was then replaced with 70% ethanol for 672 hrs. The second experiment had the same design except an additional set of cells were subjected to heat-induced AR concomitantly. PCNA, cytokeratins AE1/AE3, and EGFr (membrane and cytoplasmic) were used to evaluate the effects of immunorecognition. Fixation in 10% NBF for 12 hrs plus transfer to 70% ethanol for 672 hrs did not preserve immunorecognition of PCNA adequately in either cell lines. Cytokeratins immunoreactivity was preserved by transfer to 70% ethanol. Cytoplasmic EGFr antigens were not adversely affected by 10% NBF fixation in either cell line and transfer to 70% ethanol had limited effects. With AR, there was little recovery of PCNA immunorecognition on cells fixed in only 10% NBF, but almost complete recovery for cells transferred to 70% ethanol. For cytokeratins there was complete recovery of immunorecognition either with only 10% NBF or 12 hrs plus transfer to 70% ethanol. For EGFr, AR resulted in complete loss of immunorecognition following either treatment. This study indicated that 12 hrs of fixation in 10% NBF plus transfer to 70% ethanol for 4 weeks with AR resulted in recovery of immunorecognition for PCNA and cytokeratins, but standard methods of AR caused loss of immunorecognition of EGFr.     

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.     

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.     

Zinc Chromate Modification of the Golgi Technic

A modification of the Golgi technic is described in which the reaction takes place in well fixed formalin material. Thin slices (whole sections of adult monkey, cat and rat cerebrum) 2 to 3 mm. thick, from brains fixed 3 to 4 months in 10% formalin, are chromated for two days in 3 g. of zinc chromate dissolved in 98 ml. of distilled water and 2 ml. of formic acid. Slices are then removed, blotted dry and immersed, suspended by a thread, in 0.75% silver nitrate solution for two days. Solution should be changed after the first day. After silvering, the slices are dehydrated rapidly (total time about one hour) in 95% and absolute alcohol, placed in xylene 10 minutes, in low melting point paraffin 10 minutes and embedded in low melting point paraffin. Only surface infiltration is necessary since sections are cut 90 to 100 u. Sections are collected in 95% alcohol, dehydrated in absolute alcohol, cleared in several changes of xylene and mounted in Fisher's Permount. Results with fetal and new born material were not good.     

Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen.

Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.     

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Staining Myelin in Brain with Gallein : A New Method

A procedure is described in which gallein, mordant violet 25, C.I. 45445, is used to demonstrate myelinated nerve fibers in animal brain. Specimens are fixed in 10% neutral buffered formalin and processed in a routine manner. Microsections are stained in an iron gallein solution with subsequent differentiation in 0.25% oxalic acid and 0.1% sodium carbonate solutions that avoid overdifferentiation. Methyl green is used to demonstrate other tissue elements. Myelin is stained deep violet, as are erythrocytes, with neuronal cell bodies and microglia shades of green. the staining procedure requires 30 minutes.