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.     

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.     

Alcoholic fixation over formalin fixation: A new,safer option for morphologic and molecular analysis of tissues

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

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.     

Tissue Fixation for Immunohistochemical Detection of Proliferating Cell Nuclear Antigen with PC10 Monoclonal Antibody

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Cedarwood Oil as a Clearing Agent with Acetic-Alcohol Fixatives

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3–24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

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.     

Paraffin Compression Due to the Rotary Microtome

The extent of compression of microtome sections has been studied for blocks with tissue and also blocks of clear paraffin. Thick sections are commonly compressed 15% or more, while in sections below 5 or 10 μ, compression may exceed 50%. Compensatory thickening of sections occurs. The degree of compression for various paraffin samples and for various conditions of knife edge, temperature, etc., is compared. Microscopical work, particularly where quantitative data or reconstructions are involved, is often seriously unpaired by unrecognized artifacts of sectioning. The present work indicates the magnitude of such artifacts. Compensation for distortions of sections is not easy because tissues, particularly dense tissues, may compress less than the paraffin matrix. Section corrugation is due to this inequality in compression. Absorption of water in section flattening causes some tissue readjustment, but this varies with different tissues and different fixations.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Microwave stabilization versus chemical fixation. A morphometric study in glycolmethacrylate- and paraffin-embedded tissue

Summary A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate).An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37–46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10–20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.     

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.     

Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections

Summary In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed 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.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy

Summary In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 m in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylineosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.