Antigen-antibody complex binding and cell interaction in stimulating normal rabbit lymphocytes

Complexes of antigen with specific antibody have been shown to enhance or suppress the specific antibody response in vivo. In vitro, antigen-antibody (Ag-Ab) complexes prepared in a slight antigen excess with rabbit antibody induced proliferation of unprimed rabbit lymphocytes. The Ag-Ab stimulated cells from a number of different normal lymphoid organs, including peripheral blood, bone marrow, spleen, and lymph node, but not thymus. Cells exposed to Ag-Ab for 1 hr and washed, bound Ag-Ab through Fc and complement receptors (CR), but were not induced to proliferate unless additional Ag-Ab was added. Specific antigen, which was otherwise unstimulatory, interacted with Ag-Ab-coated cells to activate them, probably through the cross-linking of membrane-bound ligands. Proliferation stimulated by Ag-Ab involved the interaction of three bone marrow cell subpopulations; a macrophage-enriched, a B-cell-enriched, and an mIgM- cell-enriched population. The separated subpopulations were poorly responsive to Ag-Ab stimulation, even though Ag-Ab bound to cells in each of the populations. Low levels of responsiveness to Ag-Ab also resulted when any two of the three subpopulations were combined. Only when all three subpopulations were mixed, was stimulation equivalent to the levels of stimulation reached by unseparated cells.     

Antigen retrieval,signal amplification and intensification in immunohistochemistry

In this overview we emphasize new methods of improving immunohistochemical results in formaldehyde-fixed tissue samples. The benefit of heat-induced antigen retrieval in demasking of concealed epitopes is demonstrated. We provide guidance on the influence of heat-induced antigen retrieval in commonly applied monoclonal and polyconal antibodies. Moreover, we show the promising methods of signal amplification using biotinylated tyramine and signal intensification of diaminobenzidine reaction products by metallic ions.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

系统解剖学立体化双语教学资源建设的探讨

系统解剖学是医学生最为重要的专业基础课程之一。近年来,我们一直坚持双语教学,2008年我校系统解剖学获得首批国家双语教学示范课程。为建设好双语教学示范课程,我们积极开展解剖学双语教学探索和实践,着重于该门课程的立体化双语教学资源的建设,以纸质教材为主体,利用不同教学媒体的优点来呈现解剖学不同的教学内容,形成一个立体化的双语教学资源,并应用于教学实践活动中,达到提高学生专业英语理解能力,灵活运用外语思维解决医学问题能力的目标。     

Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval

p53 is the most frequently altered tumor-suppressor gene in skin cancer. In normal tissues the p53 protein (wild type) has a very short half-life and it is not detectable immunohistochemically. In contrast, the mutant p53 protein has an extended half-life in tumor cells and can be detected by immunohistochemical methods. p53 is widely used as an indicator of tumor aggression and progression. Fixation methods especially formaldehyde based fixation may mask the immunohistochemical detection of p53 protein but antigen retrieval methods enhance the inmmunohistochemical detection of p53 protein by remodification of protein structure. This study was designed to evaluate the efficacy of different fixatives, of microwaving and microwave pretreatment method to retrieve p53 immunoreactivity in paraffin-embedded non-lesioned (adjacent normal tissue) human skin samples or pathological human skin samples diagnosed as basal cell carcinoma. The samples were fixed at RT and/or in microwave oven either in neutral buffered formalin or alcohol for different time periods. For antigen retrieval, the sections were irradiated in a microwave oven for 5 cycles in 10 mM citrate buffer (pH 6.00). In this study the effects of six different fixation methods on the immunohistochemical staining have been investigated in basal cell tumor specimens. The application of antigen retrieval method was also examined and compared. Optimal results were obtained using samples fixed in alcohol either at room temperature (24 h) or in microwave oven.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

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.     

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Abstract

The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.     

A new antigen retrieval technique for human brain tissue

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.     

The expression of hepatitis-C virus antigen by immunohistochemical stain and polymerase chain reaction in corneas of seropositive corneal donors

Background: Of the donor corneas rejected for transplantation, the largest group is that from donors testing seropositive for hepatitis C virus (HCV). In situations of severe shortage in supply of donor corneal tissue, we may consider the use of seropositive donors for transplantation if we can prove with high certainty the absence of HCV RNA in the donor corneal tissue. Polymerase chain reaction (PCR) is a highly sensitive and specific technique for direct detection of HCV RNA and can be used for this purpose. Nevertheless, it is not applicable for routine clinical use in most eye departments due to its unavailability and cost effectiveness.Purpose: To study the possible use of immunohistochemical method for detection of HCV antigen in corneal tissue of seropositive donors and correlate the results with those of PCR. Immunohistochemical methods have not yet been studied in donor corneal tissue.Materials and methods: Eight corneas of 4 seropositive and 8 corneas of 4 seronegative corneal donors were studied by immunohistochemical and PCR methods for the presence of HCV antigen in their corneal tissue and sera.Results: HCV RNA was not detected in the sera and corneal tissue of all seropositive and seronegative corneal donors by either PCR and immunohistochemical methods.Conclusion: Although the study is too small for conclusive results, the correlation between the immunohistochemical and PCR studies for direct detection of HCV antigen in corneal tissue of seropositive donors may raise the possibility of using the immunohistochemical method for screening of donor corneas for the detection of HCV antigen. A larger prospective study investigating the sensitivity, specificity and clinical applicability of the immunohistochemical method is warranted. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Serological Comparison of Selected Parasitic and Free-Living Amoebae in vitro,Using Diffusion-Precipitation and Fluorescent-Antibody Technics*

SYNOPSIS. The present study has been directed to a serological comparison of three closely similar species of Entamoeba: E. histolytica, E. invadens and E. moshkovskii, and two free-living soil amoebae: Hartmannella rhysodes and Mayorella palestinensis. Except for E. histolytica and E. moshkovskii, the other amoebae used here were grown axenically; this is the first report of the use of antigenic extracts from axenic cultures of parasitic amoebae. The method described here provides a potent antigen that elicits a good antibody titer and is generally applicable to both parasitic and free-living amoebae. Amoebae pooled from well-grown cultures were thoroughly washed, sonicated and mixed with Freund's Adjuvant; this antigenic preparation was injected into rabbits. Two subcutaneous injections were given at three-week intervals, and 2-3 weeks thereafter blood was withdrawn to obtain antiserum. Agar-gel diffusion, cellulose acetate paper and fluorescent antibody technics were used to test the antigen-antibody (Ag-Ab) reactions. Results of the Ag-Ab reactions can be summarized as follows: 1) The homologous Ag-Ab reaction was obtained in all cases tested. 2) There was no serological reaction between the parasitic and free-living amoebae tested. 3) There was a definite serological reaction between H. rhysodes and M. palestinensis. 4) Multiple antigens were found in E. invadens (PZ strain) and E. histolytica (DKB strain) when they were tested against anti-PZ serum and anti-DKB serum, respectively, and no reaction was found when the other test antigens were exposed to these two antisera in gel-diffusion tests. 5) With the fluorescent antibody technic, E. histolytica (Laredo strain), E. moshkovskii (DSR strain) and E. histolytica (DKB strain) showed some degree of serological reaction in descending order when they were stained with conjugated anti-E. invadens serum.     

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.     

The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin–biotin–peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.     

一种改良的BrdU免疫组织化学染色抗原热修复技术

目的：探索一种简单有效的行BrdU免疫组织化学染色抗原热修复的方法。方法：本实验探索了三种抗原热修复方法：1）漂片煮沸法,2）贴片煮沸法,3）贴片改良法。将贴有冰冻组织切片的玻片置于恒温封闭体系中进行热修复。之后行BrdU免疫组化染色,栗集图像对这三种热修复方法进行比较。针对贴片改良法行BrdU与NeuN、P。CERB和DCX的荧光双标,采集图像以观察该方法在免疫荧光甄标实验中应用的可行性。结果：1）漂片煮沸法脑组织切片在经过热修复处理后,切片卷起皱缩,不能进行后续实验步骤;2）贴片煮沸法可见少量BrdU阳性细胞,但背景深,且少数脑片起泡,假阳性现象严重;3）贴片改良法B-U免疫组化及与NeuN、P-CERB和DCX的免疫荧光双标染色背景浅,阳性细胞明显。结论：采用改良的热修复法能充分暴露BrdU抗原,DAB显色及免疫荧光染色结果理想。此方法为一种较好的行BrdU免疫组织化学染色抗原热修复方法。     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Combining Immunodetection with Histochemical Techniques: The Effect of Heat-induced Antigen Retrieval on Picro-Sirius Red Staining

Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.