A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

改良的Cajal金升汞染色法显示纤维性星形胶质细胞

目的探索一种快捷、简便、稳定的染色方法显示脑组织内纤维性星形胶质细胞。方法取猫、家兔、大鼠和豚鼠的大脑组织,部分组织采用传统的Cajal金升汞法制片。部分组织采用改良Cajal金升汞法制片。结果与传统Cajal金升汞法比,应用改良Cajal金升汞染色法,缩短了制片时间,纤维性星形胶质细胞染色均匀,结构清晰,胶质细胞纤维显现明显,分枝光滑,较长突起末端膨大的脚板终止于血管壁上。结论应用改良后的Cajal金升汞法染色显示纤维性星形胶质细胞效果明显优于传统Cajal金升汞法。     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)     

Immunogold-silver cytochemistry using a capillary action staining system

An improved method for immunogold-silver staining is described. Rat monoclonal antibodies to T-lymphocyte surface membrane antigens were employed to demonstrate T-cells and their subsets in frozen sections of mouse spleen and in smears of peripheral blood mononuclear cells. A capillary action staining system was used to perform all stages of the procedure, substantially reducing the technical effort involved, saving reagents, and improving reproducibility and control of batch staining. The use of a light-stable silver enhancement reagent simplified the development of the reaction product. Technical modifications were incorporated to obtain good preservation of tissue morphology when employing this reagent on frozen sections and to stabilize the reaction product. The improved immunostaining procedure was rapid, sensitive, and yielded better staining quality, with excellent contrast and minimal nonspecific precipitation of silver.     

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。     

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。     

Histochemical localization of glutathione in tissues.

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.     

Effects of primary antiserum dilution on staining of "antigenrich" tissues with the peroxidase antiperoxidase technique.

The effect of primary antiserum dilution on staining results with the peroxidase antiperoxidase method were investigated using frozen sections of perfused rat cerebellum and optic nerve. Results comparable to formalin fixed and paraffin embedded tissue were attainable only when low antiserum concentrations were used. Optimal staining of antigen rich tissue, such as frozen sections, with the peroxidase antiperoxidase method required low antiserum concentrations apparently to minimize the binding of both antigen-binding fragments of the bridging antibody to the tissue bound antiserum. It appears that low antiserum concentration insures that sufficient bridge antibody molecules will be only singly bound and thus free to attach the peroxidase antiperoxidase complex.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

A rapid, sensitive histochemical stain for myelin in frozen brain sections.

A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.     

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

Wheat Germ Agglutinin Staining as a Suitable Method for Detection and Quantification of Fibrosis in Cardiac Tissue after Myocardial Infarction

The quantification of fibrotic tissue is an important task in the analysis of cardiac remodeling. The use of established fibrosis staining techniques is limited on frozen cardiac tissue sections due to a reduced color contrast compared to paraffin embedded sections. We therefore used FITC-labeled wheat germ agglutinin (WGA), which marks fibrotic tissue in comparable quality as the established picrosirius red (SR) staining, for the staining of post myocardial infarction scar tissue. The fibrosis amount was quantified in a histogram-based approach using the non-commercial image processing program ImageJ. Our results clearly demonstrate that WGA-FITC is a suitable marker for cardiac fibrosis in frozen tissue sections. In combination with the histogram-based analysis, this new quantification approach is i) easy and fast to perform; ii) suitable for raw frozen tissue sections; and iii) allows the use of additional antibodies in co-immunostaining.Key words: Cardiac fibrosis, myocardial infarction, wheat germ agglutinin, fibrosis quantification, histogram-based analysis     

Tissue distribution and ultrastructure of B lymphocytes reacting with the monoclonal antibody anti-Y29/55

The reactivity of normal tonsilar cells with the monoclonal antibody anti-Y29/55 is characterized at the tissue and ultrastructural cytological level. Using an indirect immuno-alkaline phosphatase method on frozen sections the antibody labels mantle zone and germinal center lymphocytes. This staining reaction is more generalized in B-lymphocyte areas than that obtained with antibodies to IgM and IgD. By indirect immunoperoxidase staining, as well as by an indirect rosetting procedure in cell suspensions, the reactive cell population were either small resting lymphocytes or activated lymphocytes corresponding to centrocytes, centroblasts, immunoblasts and plasmoblasts; some plasma cells were also labeled. These results characterize the monoclonal antibody anti-Y29/55 as a pan-B-marker antibody, useful for labeling resting and activated peripheral B-lymphocytes in frozen tissue sections and cell suspensions.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.     

Investigation of Tissue Preparation Conditions for Non-radioactive In Situ Hybridization: Localization of Transforming Growth Factor-α Message in Rat Kidney

A non-radioactive method of in situ hybridization was used to localize transforming growth factor- mRNA in epithelial cells of collecting ducts and tubules in rat kidney tissue sections. The intensity and specificity of staining were assessed under a variety of tissue preparation conditions, including a direct comparison of paraffin against frozen sections. Under optimal conditions, both the signal strength and the cellular localization of the growth factor message were superior in paraffin sections. The staining method could also be used to localize the message in lung tissue, indicating that the procedure is generally applicable to other tissues. Our results indicate that the use of paraffin sections for non-radioactive in situ hybridization affords a number of advantages for the localization of specific messages in tissue sections.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.