[Tissue fixation of spiramycin in guinea pig; comparison with other antibiotics].
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Anti-Bacterial Agents↗
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Effects of tissue fixation and processing on proliferating cell nuclear antigen (PCNA) immunohistochemistry.
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Animals↗
The effect of tissue fixation on immunostaining for proliferating cell nuclear antigen with the monoclonal antibody PC10.
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Antibodies, Monoclonal↗
A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples.
Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.
Animals↗
Effect of tissue fixation on recovery of DNA adducts in the 32P-postlabelling assay.
The suitability of fixed tissue as a source of DNA for 32P-postlabelling studies on DNA adduct formation has been investigated. Tissues (spleen, liver, lung, colon and kidney) from rats treated i.p. with benzo[a]pyrene (BaP) (100 mg/kg) or 2-acetylaminofluorene (AAF) (40 mg/kg) were removed and placed in formalin fixative for 1, 7, 28 or 92 days. DNA was isolated and 32P-postlabelled with nuclease P1 enhancement for BaP-modified DNA and with butanol enhancement for AAF-modified DNA. There was a marked loss of adducts with time of fixation, most noticeably between 1 and 7 days. DNA from tissue that had been fixed for 92 days and then wax-embedded had similar levels of adducts to the 92-day fixed-only samples. Further tissue samples were fixed for 24 h with formalin, modified Methacarn or Bouin's and then wax-embedded. DNA was then extracted either immediately or 28 days later. Adducts were recovered from formalin- and Methacarn-treated tissues at levels comparable to those detected in DNA from frozen tissues and levels were stable once the tissues were wax-embedded. However, DNA recovery from Bouin's-fixed tissue was poor. The results indicate that only tissues that have been wax-embedded after the minimum required fixation period are suitable sources of DNA for 32P-postlabelling analysis.
2-Acetylaminofluorene↗
The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution.
We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoy-fixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some beta-oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.
Acetates↗
Effects of tissue fixation conditions and protease pretreatment on immunohistochemical performance of a large series of new anti-keratin monoclonal antibodies: value in oncopathology.
A comparative study with 21 recently raised monoclonal antibodies (3 of which are reported here for the first time) to human keratin polypeptides was performed on a wide range of paraffin-embedded tissues and tumors, aimed at the examination of effects of four different fixatives and protease pretreatment on the immunohistochemical detection of keratins. Our data demonstrated that: (a) formaldehyde-based fixatives modified by acidification and/or addition of methanol gave results superior to those achieved by routinely used formol saline; (b) relatively rare antibodies (4 out of 21) could be identified which gave reliable immunostaining patterns even on routine formalin-fixed material; (c) a proteolytic digestion step preceding the immunostaining was beneficial for the performance of the majority of antibodies in our panel. Additional options which could potentially lead to further improvement of keratin immunohistochemistry in paraffin embedded specimens are also suggested. This work provides the necessary basis for wider application of the anti-keratin antibodies of the C-series in both routine oncopathology and research-oriented retrospective studies.
Adenocarcinoma↗
Microwave oven for improved tissue fixation and decalcification.
The present study reports on our experience on microwave (MW) fixation and decalcification. A common kitchen microwave oven was used. Autoptical material and biopsies from different organs were immersed in 10% formalin and MW irradiated varying time and power. Bone biopsies were immersed in 5% formic acid and MW irradiated. The tissues were then routinely embedded in paraffin. The results, checked on routinely stained sections, indicate that MW irradiation speeds up both fixation and decalcification. Optimal treatment conditions are indicated.
Decalcification Technique↗
Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry.
We investigated the influence of various fixatives on monoclonal anti-BrdUrd antibody binding of BrdUrd-substituted DNA in tissue sections of routinely processed mouse small intestine after in vivo administration of BrdUrd. For denaturing fixatives such as ethanol or Carnoy's fluid, a standard denaturation protocol showed specific crypt cell labeling. With cross-linking agents such as formalin and glutaraldehyde, a remarkable increase in staining intensity was obtained after tissue digestion with pepsin before acid denaturation. The optimal pepsin concentration was determined for maximal immunoreactivity combined with acceptable morphology.
Animals↗
Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds.
Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (approximately 0.25%) considerably less concentrated with respect to OsO4 than solutions (1-2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry. These results suggest that the effectiveness of OsO4 as a fixative may, in part, be related to its nonpolarity. The infrared spectra indicate that the OsO4 molecule is tetrahedral, perfectly symmetrical and, therefore, as a whole nonpolar. As a consequence, it could be expected to readily penetrate charged surfaces of tissues, cells, and organelles. The spectral studies show that osmeth is much less symmetrical and, to that extent, polar; thus, it penetrates biomembranes less readily.
Animals↗
Immunocytochemical localization of Fos in perfused nonhuman primate brain tissue: fixation and antisera selection.
Immunocytochemical localization of immediate early gene proteins, such as Fos, provides a powerful tool with which to demonstrate activated neuronal populations in response to specific stimuli. In contrast to studies using rat brain tissue that consistently show good Fos detection with a variety of antisera, studies using brain tissue from other species yield variable Fos detection. This may be partly due to differences in Fos protein sequences among species or to perfusion and fixation methods. To determine the ability of various Fos antisera to detect neuronal activation in nonhuman primate tissue, we tested nine Fos antisera and compared these antibodies under conditions of intense or physiological stimulation. Monkey brain tissue was either perfused and postfixed with 4% paraformaldehyde or perfused with 4% paraformaldehyde and postfixed with 2.5% acrolein in 4% paraformaldehyde. In rat tissue, stained for comparison, several antisera resulted in good to excellent Fos detection. However, few antisera tested in monkey tissue resulted in excellent Fos staining. We demonstrate that detection of Fos in monkey brain tissue perfused with 4% paraformaldehyde can be improved by postfixation in a dilute acrolein solution. Our findings emphasize the importance of choosing appropriate antisera and perfusion-fixation procedures to optimize Fos detection in nonhuman primate tissue.
Acrolein↗
Tissue fixation with phenol-formaldehyde for routine histopathology.
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 (pH 7.0) 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 pH 7.0 and pH 5.5 was carried out at an elevated temperature (40 degrees C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol-formaldehyde (pH 7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol-formaldehyde (pH 7.0) 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 (pH 7.0) 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 (pH 7.0) and neutral buffered 4% formaldehyde showed similar volume changes.
Animals↗
Silver-stained structures (AgNORs), their dependence on tissue fixation and absence of prognostic relevance in rectal adenocarcinoma.
The value of a one-stage silver staining technique for nucleolar organizer region (NOR)-associated protein was investigated as a predictor of clinical outcome in 100 rectal adenocarcinomas. In formalin-fixed tissues, a variety of silver-stained structures ranging from 0.5 to 7 microns are seen, the abundance of which bears no relation to prognosis, cell proliferation, or ploidy. Evidence is presented that the silver-stained structures are the result of coalescence of smaller particles caused by formalin fixation, and that assessment of NOR activity is not reliable in routinely formalin-fixed archival tissues.
Adenocarcinoma↗
Limited tissue fixation times and whole genomic amplification do not impact array CGH profiles.
BACKGROUND: Array comparative genomic hybridisation (CGH) is a powerful method for the genetic analysis of lesional and normal tissues to identify genomic imbalances associated with malignancies. However, the use of this technique with DNA extracted from archival formalin fixed, paraffin embedded (FFPE) tissue specimens, the most widely available resource for retrospective studies, is subject to quantitative and qualitative limitations. In this report, the suitability and integrity of the DNA extracted from FFPE MCF7 breast cancer cells fixed for different periods of time for array CGH applications were examined. RESULTS: Using our established cDNA microarray protocol in conjunction with whole genome amplification methods, the genetic profiles of freshly harvested MCF7 cells and their matched FFPE counterparts were analysed. Congruent profiles between FFPE MCF7 cells and their fresh counterpart and between amplified and non-amplified FFPE MCF7 cells were observed. Our results demonstrate that formalin fixation of <20 hours has no significant adverse effect on the integrity of DNA for array CGH studies. CONCLUSIONS: Our findings attest to the fidelity of our array CGH methods to effectively examine material recovered from FFPE tissue specimens for microarray applications. This in turn has great potential to identify novel diagnostic and prognostic markers for human disease.
Breast Neoplasms↗
Killing of Mycobacterium tuberculosis in tissue by microwaves with simultaneous tissue fixation.
Guinea-pig liver heavily infected with M. tuberculosis has been sterilised by exposure to microwaves in a standard commercially available domestic oven. Subsequent histology showed good tissue preservation and organisms of normal morphology were identified by Ziehl Neelsen staining. The findings allow the safe use of frozen sections for diagnosis in tissue containing M. tuberculosis.
Animals↗
Effect of tissue fixatives on telomere length determination by quantitative PCR.
Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.
Cellular Senescence↗
On velocity changes caused by tissue fixation.
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Formaldehyde↗
Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact.
The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.
Animals↗