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.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

Histological assessment of PAXgene tissue fixation and stabilization reagents

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Methods for decreasing interstitial immunoglobulin in tissue slices and cryostat sections

To determine whether lymphoid antigens and cellular morphology can be preserved after long-distance transport in buffer or cell culture medium, we stained cryostat sections prepared from human tonsil samples that had been kept at 4 degrees C or 20 degrees C for 24, 48 or 72 h. B-Cell antigens, T-cell antigens, and Ia antigens were well preserved after storage up to 72 h in buffer or medium at 4 degrees C. Interstitial immunoglobulin (Ig) was decreased following all incubation procedures. We then investigated methods to diminish interstitial Ig in cryostat sections, since it would be inconvenient to keep 2-3 mm tissue slices in buffer or medium prior to freezing and subsequent Ig staining. Cryostat sections were air dried or briefly fixed in acetone prior to washing in buffer or medium at 4 degrees C, 20 degrees C or 37 degrees C for 1, 2 or 24 h. Then sections were air dried or washed prior to acetone fixation and immunostaining. A method for washing cryostat sections was developed which diminished interstitial Ig without compromising the quality of immunostaining or cellular detail. These methods are especially useful for studying samples of lymphoid tissue in which the presence of large quantities of interstitial Ig obscures the detection of monotypic Ig staining patterns.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Intragranular colocalization of immunoreactive methionine-enkephalin and oxytocin within the nerve terminals in the posterior pituitary

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.     

Improvements in histological techniques for epoxy-resin embedded bone specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 x 3.0 cm. Mineralized sections are cut to 4 micrometer; demineralized at 3 micrometer. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time–dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins:     

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.     

Application of Microwave Irradiation to Immunohistochemistry: Preservation of Antigens of the Extracellular Matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodatelysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)     

Enzyme, Lectin and Immunohistochemistry of Plastic Embedded Undecalcified Bone and other Hard Tissues for Light Microscopic Investigations

Enzymes and tissue antigens were localized on plastic embedded undecalcified bones and teeth using Technovit 7200 VLC (Kulzer, Germany). This resin is hard enough for cutting and grinding procedures on rotating plates with diamond layers. The pores between the diamond grains are not obstructed with this resin. The procedure described here permits localization of antigens in the soft tissues adjacent to, or in the biological hard tissues themselves and in dental implants (ceramic or metallic) on the light microscopic level. The undecalcified bone is fixed and embedded in plastic and cut at 100-150 μm. The slices are ground automatically by a grinding machine to a thickness of 5-10 μm. After application of the substrates for alkaline and acid phosphatases and the required dyes, the distribution of these enzymes can be demonstrated. Tissue antigens also can be detected with slightly modified standard techniques of immunohistochemistry and lectin histochemistry using the peroxidase technique or fluorescence microscopy.     

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.     

Generation of high‐quality protein extracts from formalin‐fixed,paraffin‐embedded tissues

A wealth of information on proteins involved in many aspects of disease is encased within formalin‐fixed paraffin‐embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this “hidden treasure” is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full‐length proteins. Here, we describe an optimised method for extraction of full‐length proteins from FFPE tissues. This method builds on the classical “antigen retrieval” technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 μg and 86.8 μg of proteins were obtained per 80 mm² tissue slice of formalin‐fixed paraffin‐embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS‐PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC‐nanoESI‐Q‐TOF MS of FFPE proteins resolved by SDS‐PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.