Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

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.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

Quantification of in situ hybridization signals in rat testes.

We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouin's fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through "posterization" of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections.     

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.     

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.     

In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.     

Detection of different mRnas expressed in the thyro-parathyroid complex of the rat by in situ hybridization using digoxigenin-labelled oligonucleotide probes

The effects have been examined of different methods and regimens for tissue fixation, preservation, permeabilization and immunostaining of different mRNAs detected by in situ hybridization in paraffin-embedded samples. The three main hormone mRNAs expressed in the thyro–parathyroid glands, namely thyroglobulin, calcitonin and parathyroid hormone mRNAs, were chosen as the target nucleic acid sequences to be detected using digoxigenin-labelled probes. Our results suggest that chemical fixation and permeabilization of tissue samples are restrictive steps. Thus, paraformaldehyde fixation provides excellent signal intensities and non-detectable background levels whereas routine formalin and Bouin's solution give unsatisfactory results. A clear linear correlation was also found between signal intensity and proteinase K permeabilization. Moreover, the optimization of immunohistochemical steps, such as anti-digoxigenin antibody concentration and colour development times, enhance the intensity and specificity of hybrid signals. Furthermore, our results show that, in contrast to some data in the literature, paraffin-embedded tissue is suitable for detection of mRNAs by in situ hybridization. It gives equivalent intensities of specific signal and superior histological and cellular resolutions when compared to cryopreserved tissue.     

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.     

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.     

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.     

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.     

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.     

Alkaline fixation drastically improves the signal of in situ hybridization

In situ hybridization (ISH) is widely used to detect DNA and RNA sequences within the cell and tissue sections. The important step in performing this technique is tissue fixation. We investigated the influence of the pH of the fixative on the outcome of ISH. Our studies indicate that alkaline formaldehyde dramatically increases the ISH signal with RNA probes. The increase in signal was observed for detection of low as well as for high abundance messages. The sensitivity of the method was increased 5- to 6-fold.     

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.     

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation