A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Controls for Immunocytochemistry: An Update

Immunocytochemistry is a highly productive method in biomedical research used to identify proteins and other macromolecules in tissues and cells. Control samples are required to show label localization is correct, but the understanding and use of immunocytochemistry controls have been inconsistent. A new classification of immunocytochemical controls is proposed that will help in understanding this most important component of the experiment. The three types of controls required for immunocytochemistry are primary antibody controls that show the specificity of the primary antibody binding to the antigen, secondary antibody controls that show the label is specific to the primary antibody, and label controls that show the labeling is the result of the label added and not the result of endogenous labeling. Publications containing immunocytochemical results must give details of how these controls were performed.     

A guide to the perplexed on the specificity of antibodies

Many investigators are unaware of the potential problems with specificity of antibodies and the need to document antibody characterization meticulously for each antibody that is used. In this review, I consider the principles of antibody action and how they define a set of rules for what information should be obtained by the investigator before using an antibody in a serious scientific investigation.     

Special symposium: fixation and tissue processing models

Abstract

Fixation and processing of tissue to paraffin blocks permit thin (4-5 µm) sections of tissues to be cut. Tissues and their subcellular components and surrounding stroma are visualized by cutting thin sections and staining them histochemically or immunohistochemically and viewing the sections using a bright field microscope. During the last century, anatomists and pathologists have used fixation with 10% neutral buffered formalin (10% NBF) as the fixative of choice. Also, both human and veterinary pathologists have trained to use fixation with 10% NBF, so these professionals are reluctant to change the familiar microscopic appearance of diagnostic tissues by using different fixatives. In addition, the effects of tissue processing on the microscopic appearance of tissue essentially has been ignored in most studies. Archives of paraffin blocks of pathological tissue contain essentially paraffin blocks fixed in 10% NBF. Therefore, if retrospective studies use archival paraffin blocks to correlate the molecular features of diseases with their outcomes, the studies must be based on tissue fixed in 10% NBF. Studies of how fixation in 10% NBF interacts with histochemical and immunohistochemical staining are limited in number and most are based on relatively long fixation times (≥36 h). Currently, fixation times in 10% NBF have been reduced to <24 h. Little is known about fixation in 10% NBF and its interaction with tissue processing for any period of fixation, especially short times. Less is known about how fixation of tissues with 10% NBF interacts with more modern assays using immunohistochemistry, real time quantitative polymerise chain reaction (PCR), and techniques that depend on analysis of proteins extracted from paraffin blocks including multiplex immunoassays or mass spectrometry. In general, multiple antibody–antigen combinations are reported not to work in tissues fixed in 10% NBF, i.e., loss of immunorecognition is nearly complete for such antibody–antigen combinations as Ki67/MIB, estrogen receptor alpha (ERα) and Progesterone receptor (PR), and partial for Bcl-2. Several models have been developed to study the interactions of tissue fixation and immunorecognition, but most have viewed the problem with immunorecognition as completely caused by fixation. Also, some of the models discussed in this special symposium do not predict the effects of fixation on frozen tissues fixed in 10% NBF and not processed to paraffin blocks. This article is a brief review of issues attending the use of 10% NBF combined with tissue processing as an interrelated process to study biomarkers identified by immunohistochemistry.     

Immunogold labeling of cryosections from high-pressure frozen cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO₄) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

Macromolecular changes caused by formalin fixation and antigen retrieval

Although the mechanics of formalin fixation and antigen retrieval have been studied extensively and reviewed periodically, little attention has been directed toward conformational changes in target molecules. Formaldehyde changes the shape of tissue molecules by appending small hydroxymethyl groups to them. These adducts, in turn, can react with other tissue molecules to form crosslinks, or they can participate in a variety of reactions during tissue processing, including formation of imines, ethoxymethyl adducts, and further crosslinks. Under the influence of alcohol dehydration, fixed DNA may fragment and form a variety of depurination products. The situation becomes even more complex with short fixation times because under these conditions, the dehydrating agent used for tissue processing denatures macromolecules in other ways, most notably through rearrangement of molecular shape to move hydrophobic realms outward and hydrophilic areas inward (hydrophobic inversions). How tissue molecules are modified affects the outcome of immunohistochemical staining and prospects for restoration of antigenicity. Immunoreacitivity may be compromised because epitopes are either sterically hidden, but otherwise unaffected, or they have been altered more directly. Enzyme-based retrieval methods are best suited for the former because they literally snip the molecule apart to reveal the portions of interest. Heat-induced retrieval with buffers can demodify affected epitopes by removing adducts and breaking crosslinks. The choice of temperature and pH is usually critical for optimal retrieval. Effective temperatures are directly related to the strength of bonds-higher temperatures are needed to break stronger bonds. The pH of the retrieval solution determines the charge on the tissue molecule; the goal is to create a charge that causes the demodified molecule to assume a near natural conformation. Rational use of these concepts should lead to better control of immunohistochemical reactions.     

Macromolecular changes caused by formalin fixation and antigen retrieval

Although the mechanics of formalin fixation and antigen retrieval have been studied extensively and reviewed periodically, little attention has been directed toward conformational changes in target molecules. Formaldehyde changes the shape of tissue molecules by appending small hydroxymethyl groups to them. These adducts, in turn, can react with other tissue molecules to form crosslinks, or they can participate in a variety of reactions during tissue processing, including formation of imines, ethoxymethyl adducts, and further crosslinks. Under the influence of alcohol dehydration, fixed DNA may fragment and form a variety of depurination products. The situation becomes even more complex with short fixation times because under these conditions, the dehydrating agent used for tissue processing denatures macromolecules in other ways, most notably through rearrangement of molecular shape to move hydrophobic realms outward and hydrophilic areas inward (hydrophobic inversions). How tissue molecules are modified affects the outcome of immunohistochemical staining and prospects for restoration of antigenicity. Immunoreacitivity may be compromised because epitopes are either sterically hidden, but otherwise unaffected, or they have been altered more directly. Enzyme-based retrieval methods are best suited for the former because they literally snip the molecule apart to reveal the portions of interest. Heat-induced retrieval with buffers can demodify affected epitopes by removing adducts and breaking crosslinks. The choice of temperature and pH is usually critical for optimal retrieval. Effective temperatures are directly related to the strength of bonds-higher temperatures are needed to break stronger bonds. The pH of the retrieval solution determines the charge on the tissue molecule; the goal is to create a charge that causes the demodified molecule to assume a near natural conformation. Rational use of these concepts should lead to better control of immunohistochemical reactions.     

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.     

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Effects of fixation for immunohistochemistry on tissue concentrations of substance P and somatostatin determined by radioimmunoassay

Concentrations of substance P and somatostatin were measured in preparations of the myenteric plexus (plus longitudinal muscle) of the guinea-pig ileum after fixation and processing for immunohistochemistry and compared with concentrations measured in fresh tissue. Two fixative solutions were used: (i) 4% formalin in phosphate buffer (0.1 M, pH 7.0); and (ii) a mixture of aqueous picric acid with 2% formalin in phosphate buffer (0.1 M, pH 7.0). Tissues were extracted in boiling aqueous acetic acid (2.0 M) either immediately after fixation and processing or after storage for up to four weeks in phosphate-buffered saline (PBS) with or without sodium azide. The concentrations of substance P and somatostatin in these extracts were measured by radioimmunoassay and compared to the concentrations in extracts of fresh tissue. The concentration of substance P in fixed tissue was the same as that found in fresh tissue, whereas the concentration of somatostatin in fixed tissue was half that found in fresh tissue (P<0.01). If the tissue was not subjected to the extensive washing for immunohistochemistry, somatostatin concentrations in fresh and fixed tissue were not significantly different. The concentration of substance P did not change on storage of the fixed tissue in PBS, either with or without sodium azide. The concentration of somatostatin decreased on storage of the fixed tissue in PBS over four weeks to 40% of its original value, but the presence of sodium azide maintained the concentration at 60% at four weeks. Neither fixative solution interfered with the radioimmunoassay except at very high concentrations. Fixation for 24h gave the highest estimates of each of the peptides. It is concluded that fixation can be a useful alternative to freezing for preservation of peptides in tissue for radioimmunoassay.     

Enzymatic HCO 3 − fixation: A common mechanism for all enzymes involved?

Conclusion Present evidence is highly suggestive of a stepwise mechanism with a common initial step of HCO ₃ ^– activation, for all enzymes that use HCO ₃ ^– rather than CO₂ as the substrate. This activated form of HCO ₃ ^– appears to be the carbonic-phosphoric anhydride, although this has only been proved rigorously with carbamoyl phosphate synthetase. Further studies using isotope scrambling and other techniques will be necessary to establish conclusively that this is the case with the other enzymes.For brevity an extensive bibliographical coverage has not been attempted, and only references directly relevant to the points discussed have been included.     

Biological nitrogen fixation: A scientific perspective

The discoveries of Hellriegel and Wilfarth ended the period of controversy about the existence of biological N₂ fixation and launched a period featuring the agronomic application of the inoculation of legumes. Serious studies of the biochemistry of N₂ fixation started in the late 1920's, and defined some of the basic properties of the N₂-fixing system. Application of¹⁵N as a tracer gave definitive evidence for the role of ammonia as the key intermediate in biological N₂ fixation. It was demonstrated in the 1950's and 1960's that nitrogenase could reduce substrates other than N₂. With the achievement of consistent cell-free N₂ fixation it was possible to resolve the nitrogenase system into two proteins, electron donors, and ATP-hydrolyzing and regenerating systems. The sequence of electron transfer was established. Recently, studies of the genetics of the nitrogenase system have defined in detail how the system is assembled and controlled.     

A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

Protein fixation and antigen retrieval: chemical studies

Abstract

Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.     

Determination of optimal rehydration,fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.