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PubMed2004

Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.

RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for beta globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives' yields for various size real-time PCR amplicons. Longer amplicons (above approximately 225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.

Adult↗
PubMedDate not supplied

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Animals↗
PubMed2006

Methacarn fixation--effects of tissue processing and storage conditions on detection of mRNAs and proteins in paraffin-embedded tissues.

In this study, we examined suitable conditions for tissue fixation with methacarn and ethanol dehydration and storage of paraffin-embedded tissues (PETs) on gene expression analysis. With fixation and dehydration of rat liver tissues for up to 16 h (overnight) and 1 week, respectively, at 4 degrees C, integrity of extracted total RNAs and polypeptides did not vary, the former integrity being constantly lower than that with unfixed frozen tissue, while protein yield was slightly reduced with increasing dehydration. Retained expression levels of mRNAs and proteins were mostly unaffected by the period of fixation but slightly fluctuated with the length of dehydration. When PETs were stored for up to 12 months, integrity of both total RNAs and polypeptides was retained at 4 degrees C but reduced at room temperature. Reduced expression levels of mRNAs and proteins were also noted by storage at room temperature after 12 and 3 months, respectively. However, neither tissue processing nor storage affected variability in either mRNA or protein levels among samples. Thus, the results suggest that, for gene expression analysis, tissues can be fixed with methacarn and dehydrated for at least 1 day and 1 week, respectively, and PETs can be stored for at least 12 months, but a temperature of 4 degrees C is preferable.

Acetic Acid↗
PubMed1995

Influence of formalin fixation time and tissue processing method on immunoreactivity of monoclonal antibody PC10 for proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase-delta, has recently been proposed as a marker of proliferation that is detectable in formalin-fixed paraffin-embedded tissue. Fixation time has been known to influence protein immunoreactivity and therefore can significantly influence the results of a quantitative immunohistochemical assay. In this study, we investigate the relationship between formalin fixation time and immunoreactivity for PCNA in paraffin-embedded sections and examine the effect of postfixation tissue treatment with modified Bouin's solution. Samples of small and large intestine from two freshly sacrificed rats were fixed in 10% buffered formalin for 6, 30, 54, 174, 340, and 508 h. Standard histological processing was performed on paired specimens whose treatment differed only by predehydration immersion in a picric acid and mercuric chloride-containing solution. Paraffin sections were reacted with monoclonal antibody PC10 in a standard immunoperoxidase assay. Staining intensity for PCNA was scored on a scale of 0 to 10, and the mean number of PCNA-positive cells per crypt (10 crypts counted) was determined. No difference between animals was found. PCNA immunoreactivity was maximal in specimens fixed for 6 to 30 h, exponentially declining with longer fixation time. The rate of decline was mitigated in the treated sections. Fixation-time dependence of PCNA immunoreactivity has immediate implications for intra- and interlaboratory comparisons, especially in experimental studies in which specimens can be stored in formalin for variable times followed by batch processing. With regard to surgical pathology specimens, this study suggests that sample comparisons are valid, since routine fixation time is within the optimal rage for PCNA immunodetection.

Animals↗
PubMed1987

Rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically useful antigens.

Microwave (MW) energy permits rapid tissue fixation for light and electron microscopy but its effects on antigen preservation have not been fully evaluated. We, therefore, fixed three samples of human skin, uterus, and cervix, and two samples of human colon and breast by MW irradiation (5 to 8 seconds) during simultaneous immersion in a dilute aldehyde mixture (2% formaldehyde and 0.05% glutaraldehyde). For comparison, similar portions of each specimen were fixed in formalin. Specimens were processed routinely and embedded in paraffin for light microscopy. Sections from each specimen were stained with hematoxylin and eosin and, by immunoperoxidase techniques, for epithelial membrane antigen, leukocyte common antigen, S-100 keratin, carcinoembryonic antigen, and factor VIII-related antigen, the latter three with and without preliminary trypsinization. Colon sections were also stained for chromogranin. In all cases, light microscopic morphology was comparable for tissues fixed by the MW method and formalin-fixed specimens, as was immunostaining for epithelial membrane antigen, leukocyte common antigen, S-100 protein, and chromogranin. Formalin-fixed tissues required trypsinization for optimal detection of keratin, carcinoembryonic antigen, and factor VIII-related antigen. In contrast, trypsin-pretreatment was not necessary to demonstrate these antigens in MW-fixed specimens and, in fact, resulted in tissue digestion. We conclude that this MW fixation method provides a means for rapidly fixing tissues for immunoperoxidase staining while preserving excellent light microscopic morphology.

Antigens↗
PubMed1979

Fixation of mammalian tissues in different fixatives and its influence on the staining with methyl green-pyronin.

This investigation is a study on the role of fixation of different mammalian tissues in different fixatives as well as in 10% neutral formalin containing different metal cations and its effect on the staining with the dye-mixture, methyl green-pyronin. The results indicate variation in the colour of the nuclei in tissues fixed in the different fixatives. The possible role of the fixatives on nuclear colouration has been discussed.

Animals↗
PubMed1987

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue.

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2000

Fixation of biological tissues with a naturally occurring crosslinking agent: fixation rate and effects of pH, temperature, and initial fixative concentration.

In an attempt to overcome the cytotoxicity problem of the glutaraldehyde-fixed tissues, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. The study was intended to investigate the rate of tissue fixation by genipin. Glutaraldehyde was used as a control. In addition, the degrees of tissue fixation by genipin at different pHs (pH 4.0, pH 7. 4, pH 8.5, or pH 10.5), temperatures (4 degrees C, 25 degrees C, 37 degrees C, or 45 degrees C), and initial fixative concentrations (0.250%, 0.625%, or 1.000%) were examined. The results obtained revealed that the rate of tissue fixation by glutaraldehyde was significantly faster than that by genipin. The degree of tissue fixation by genipin may be controlled by adjusting its fixation duration or fixation conditions. The order in degree of tissue fixation by genipin at different pHs, from high to low, was: at nearly neutral pH (pH 7.4 or pH 8.5) > at basic pH (pH 10.5) > at acidic pH (pH 4.0). The degrees of tissue fixation by genipin at different temperatures were about the same, except for that at 4 degrees C. In contrast, the initial fixative concentration did not seem to affect the degree of tissue fixation by genipin, if only the amount of genipin in the fixation solution was sufficient to complete tissue fixation. The concentrations of genipin in the aqueous solutions at different pHs, temperatures, and initial fixative concentrations tended to decrease with time with or without the occurrence of tissue fixation. This indicated that genipin was not stable in the aqueous solution. The instability of aqueous genipin was more remarkable with increasing pH or temperature. The results obtained in this study may be used to optimize the fixation process for developing bioprostheses fixed by genipin.

Animals↗
PubMed1981

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.

Colon↗
PubMedDate not supplied

Use of microwaves for rapid fixation of tissues in vivo.

Accurate knowledge of the concentration in the central nervous system of neurochemicals undergoing rapid enzymatic destruction or synthesis is sparse because of the difficulty in stopping the rapid reactions while causing only minimal adverse changes in the neurochemistry and structure. Microwave heating can be effectively used to rapidly stop enzyme activity in the central nervous system with minimal adverse changes. This rapid inactivation of the enzymes increases the validity of the sample that is taken for analysis of the concentration of the enzyme's substrate.

Animals↗
PubMed1993

Rapid cold fixation of tissue samples by microwave irradiation for use in electron microscopy.

A cold microwave irradiation procedure was developed to fix rapidly and stain various tissues and monolayers for electron microscopy. Because microwave stimulation always produces some heat, melting ice was used to maintain the temperature of the tissue samples, the fixative, and the staining solution at 0 to 4 degrees C. The low temperature also reduced vapour formation, thus minimizing the risk of explosion. The microwave method shortened the total time of fixation and dehydration from the usual 3 h required by the conventional method to 65 min. After microwave fixation, the ultrastructural details of membranes and subcellular structures were excellent.

Animals↗
PubMed2004

Sodium chloride fixation of tissues under field conditions in tropical countries.

Fixation of skin and lymph node fragments in anhydric sodium chloride at room temperature for periods of weeks or months was found to preserve morphological structures and immunoreactivity. Fragments of a size of 6-8x6x6 mm were dried and placed in anhydric sodium chloride powder in sealed containers. Prior to further processing, the specimens were desalinated and rehydrated, then snap-frozen, sectioned, fixed in acetone and stained with monoclonal antibodies. The intensity of staining of molecular structures did not differ from that of primarily snap-frozen specimens. The method allows fixation of tissues under field conditions in tropical countries and at places lacking freezing facilities.

Humans↗
PubMed2005

Ultrasound-accelerated tissue fixation/processing achieves superior morphology and macromolecule integrity with storage stability.

We demonstrate that high-frequency and high-intensity ultrasound (US) can be applied to both tissue fixation and tissue processing to complete the conventional overnight formalin-fixation and paraffin-embedding (FFPE) procedures within 1 hr. US-facilitated FFPE retains superior tissue morphology and long-term room temperature storage stability than conventional FFPE. There is less alteration of protein antigenicity after US-FFPE preservation so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. US-FFPE tissues present storage stability so that room temperature storage up to 7 years does not significantly affect tissue morphology, protein antigenic properties, RNA distribution, localization, and quantitation. In addition, during fixation, tissue displays physical changes that can be monitored and reflected as changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls. The mechanism of less alteration of protein antigenicity by US-FFPE was discussed.

Autopsy↗