Detection of myelination using a novel histological probe.

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease.     

A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes.     

Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 microg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Novel DNA staining method and processing technique for the quantification of undamaged double-stranded DNA in epidermal tissue sections by PicoGreen probe staining and microspectrophotometry.

Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave-vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA.     

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。     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers

Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers can be exposed to various histochemical reactions and the staining patterns compared on the same slide to those of frozen muscle and skinned bundles. By this procedure, three fiber types were distinguished by both Ca2+-ATPase and SDH reactions. The fiber typings determined from both enzyme systems correlated well with each other. Although we were able to differentiate only between slow and fast fibers by SDS-PAGE, these results corroborated the histochemical classification. This procedure will clearly be useful in skinned single muscle fiber mechanics experiments performed to determine functional differences among fiber types.     

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.     

The use of avidin-biotin interaction in immunoenzymatic techniques.

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.     

Biochemical analysis and immunohistochemical determination of cardiac troponin for the postmortem diagnosis of myocardial damage

Cardiac disease is the most common cause of sudden death in Western nations. In forensic practice there is a need for more sensitive diagnostic methods for the postmortem diagnosis of myocardial damage. The aim of this study was to analyse the diagnostic efficacy of biochemical markers in cadaver fluids in conjunction with histological studies and the immunohistochemical determination of cardiac troponin C (cTnC) and cardiac troponin T (cTnT) levels in myocardial tissue fixed in formol and included in paraffin. We studied 50 cadavers (43 males and 7 females) with a mean age of 47.5 years (SD 19.2; range 12 to 87 years). Cases were chosen according to the postmortem interval, cause of death, and circumstances of death. Pericardial fluid and serum were tested in duplicate for cardiac troponin I (cTn I), myoglobin and CKMB by immunoassay system using commercial kits. In myocardial tissue, histological studies were performed with hematoxylin and eosin (HE), Masson's trichrome staining and immunohistochemical techniques involving streptavidin-biotin-peroxidase were performed. The results pointed to statistically significant differences for all the biochemical markers in pericardial fluid. The highest levels were obtained in the group of cadavers who had died from myocardial infarction. The immunohistochemical expression of cTnC was detected in 86% of cases; it was strongly positive and usually diffuse. The expression of cTnT, was much less frequent (46% of cases) and less intense. It was concluded that the immunohistochemical determination of cTnC and cTnT levels in myocardial tissue may be used as an index of myocardium damage.     

中国青海地区喜马拉雅旱獭嗜肝病毒自然感染的组织学研究

应用原位杂交技术、免疫组化技术以土拨鼠肝炎病毒(Woodchuck hepatitis virus,WHV)的检测系统检测50份 喜马拉雅旱獭肝组织可能存在的嗜肝病毒c基因、s抗原及c抗原的表达,同时检测肝脏组织病理学改变。结果显示 旱獭肝组织中嗜肝病毒s抗原、c抗原的阳性率分别为26％(13／50)、36％(18／50);在抗原双阳性的10份肝组织标本 中有c基因的阳性表达,阳性率为50％。c抗原定位于肝细胞胞浆和／或胞核,呈散在、片簇状分布,c基因定位于肝细 胞的细胞核,阳性细胞散在分布。50份标本中5份出现肝炎的病理改变,与抗原检出间无明显相关性。使用WHV 的病毒检测系统证实青海地区喜马拉雅旱獭可能存在类似WHV的嗜肝病毒感染,从组织学的角度为中国青海地区 喜马拉雅旱獭嗜肝病毒自然感染提供证据,此种动物有可能用于建立嗜肝病毒感染的动物模型。     

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.     

Mitochondrial abnormalities in dermatomyositis: characteristic pattern of neuropathology

The objective of the work described in this paper was to evaluate mitochondrial abnormalities in perifascicular atrophic fibers in muscle biopsies from patients with dermatomyositis (DM). We localized cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemically in muscle biopsies of 12 patients with DM, and 12 control patients with neurogenic atrophy. These two histochemical techniques were also combined on single tissue sections in order to accentuate any COX-negative fibers. Eleven out of 12 patients (91.6%) with DM showed histochemical evidence of mitochondrial dysfunction in perifascicular distribution. Similar abnormalities in histochemical staining were not seen in comparably sized myofibers that were atrophic due to denervation. It is concluded that abnormal SDH and COX histochemical activities in atrophic perifascicular fibers are characteristic of dermatomyositis. These abnormal staining characteristics could not be accounted for solely by myofiber atrophy, or by generalized abnormalities in histochemical staining.     

Immunohistochemical studies on the localization of fibronectin in human thyroid neoplastic tissues

Fibronectin (FN) has been considered to be involved in the malignant transformation of cells. It was thus of interest to morphologically study the cell surface distribution of FN in various types of thyroid neoplasms employing immunohistochemical techniques. Immunohistochemical staining using the specific anti-human FN antibody was performed by the avidin-biotin-peroxidase complex procedure. Compared to the staining pattern of normal thyroid tissues (control), differences in staining in malignant thyroid lesions and even in fetal and embryonal adenomas were evident, often on the lateral and basal aspects of the cell membrane. Colloid adenomas, however, showed FN staining on the basement membrane, basically similar to the controls. Moreover, in thyroid carcinoma, different staining patterns relating to each of the histological types were observed. The present investigation demonstrates the heterogeneity of the thyroid neoplasms in terms of FN binding.     

Ammonium Thiocyanate for Detaching Antibodies from Histological Specimens

When different antigens must be demonstrated in the same structure, the permanence of former antibodies can lead to false identification of another antigen. The peroxidase-antiperoxidase method was used, followed by the oxygen acceptor ethyl-carbazole. After staining the sections, they were destained with xylene and the antibodies detached with 3 M ammonium thiocyanate; then the specimens were treated for the demonstration of the other antigen. The procedure could be repeated and thus as many as four antigens could be demonstrated without damaging the tissues. Antigens participating in the immunohistochemical staining were well-preserved after destaining and detaching the antibodies as demonstrated by their ability to react again in a second staining.     

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.     

Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.     

扬子鳄小胃黏膜的组织化学和细胞学

扬子鳄（Alligator sinensis）的小胃是在胃幽门部与十二指肠的交接处由胃幽门括约肌和十二指肠括约肌突入管腔形成的一个小腔,其生理功能一直不清楚。本文采用组织化学、电子显微镜和免疫组织化学技术对扬子鳄小胃黏膜的组织化学成分、细胞超微结构及细胞类型进行了较全面的研究。小胃黏膜上皮PAS反应呈强阳性,AB染色呈弱阳性,主要分泌中性糖蛋白和少量的含硫酸性糖蛋白。电子显微镜下,扬子鳄小胃黏膜上皮主要由表面黏液细胞组成,偶见内分泌细胞。小胃腺部则70％-90％为内分泌细胞,其余为少量的腺黏液细胞和泌酸胃酶细胞。应用7种胃肠激素的抗血清在小胃黏膜中检测出了5-羟色胺（5-HT）、胃泌素（Gas）、胰高血糖素（Glu）、生长抑素（SS）、P-物质（SP）和血管活性肠肽（VIP）细胞,以Glu细胞密度最高,VIP细胞密度最低。未检测到胰多肽（PP）细胞。本研究结果表明,从组织化学成分和细胞类型看,扬子鳄的小胃与胃同源;从细胞超微结构和内分泌细胞所占百分比例看,扬子鳄的小胃已出现了明显的特化,泌酸胃酶细胞中未见泌酸小管,可能没有泌酸功能。内分泌细胞含量丰富,可能在调节胃肠道功能中发挥重要作用[动物学报54（6）：1044—1050,2008]。     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.