Standardized Thionin-Eosin Y: A Quick Stain for Cytology

Two stains long used in exfoliative cytology, the hematoxylin-eosin Y and Papanicolaou stains, have not been standardized even today. Some dozens of hematoxylin and eosin and Papanicolaou staining recipes have been recommended in the literature. Consequently, the staining pattern of hematoxylin and eosin, and Papanicolaou stained cytological material varies from laboratory to laboratory. To a certain degree this is due to batch-to-batch variations of commercial samples of the natural dye hematoxylin (C.I. 75290). The present paper describes a simple, standardized and reproducible procedure using thionin bromide to replace hematoxylin in the hematoxylin and eosin stain.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.     

Use of dark-field and fluorescence microscopy to examine extracellular hyaline accumulation in the mouse

Small foci of diethylstilbestrol-induced extracellular hyaline material were visualized within the mouse uterus utilizing dark-field and fluorescence microscopy following conventional hematoxylin and eosin staining. Hyaline was observed throughout the connective tissue stroma as well as between smooth muscle cells of the myometrium. The use of these techniques permits the rapid examination of small aggregates of hyaline material which are not visible with conventional hematoxylin and eosin staining and eliminates the need for time-consuming and complicated histochemical techniques. The procedures are simple and may also facilitate the quantitative (morphometric) analysis of this material.     

幽门螺杆菌感染促进胃炎儿童胃粘膜细胞的增殖

本研究旨在探讨幽门螺杆菌感染对小儿慢性胃炎患者细胞增殖的影响,使用内镜检查消化不良患者的上消化道症状,使用改良的Giemsa染色检测胃粘膜活组织中幽门螺杆菌,用苏木精/曙红和改良的吉姆萨染色活组织,并通过光学显微镜研究染色后胃粘膜样品组织病理学变化,RT-PCR检测各组胃粘膜细胞中调控细胞凋亡的Bcl-2、Bcl-xl、Bax和PCNA的mRNA表达,提取胃粘膜细胞蛋白质,利用蛋白质免疫印迹分析蛋白质浓度。组织化学染色结果表明,与对照相比,患有胃炎和幽门杆菌感染后的胃炎患者胃粘膜细胞明显增加,且幽门螺杆菌感染后细胞增殖更显著(p<0.05);幽门螺杆菌感染后Bcl-2和Bcl-xl,PCNA在患者体内表达显著上调(p<0.05),而细胞促凋亡因子Bax基因在胃炎患者感染幽门螺杆菌后被显著下调(p<0.05),蛋白免疫印迹分析Bcl-2,Bcl-xl,Bax和PCNA蛋白表达趋势与基因表达一致,说明结果可靠。幽门螺杆菌感染会显著提高慢性胃炎儿童患者胃粘膜细胞的增殖。     

Phloxine As An Histologic Stain, Especially In Combination With Hematoxylin

A staining schedule employing phloxine as a counter-stain to Erlich's acid hematoxylin is presented. Fixation is best with Zenker's fluid, although formalin can be used. The technic is similar to the standard hematoxylin-eosin formulae but because of the staining advantages of phloxine over eosin, the technic is simpler, and quicker, resulting in clearly differentiated sections which do not fade as soon as do eosin-stained slides. A brief summary of the uses of phloxine as a biological stain is given and its advantages over eosin are discussed.     

Immunofluorescence and Cytochemical Studies of Visna Virus in Cell Culture

Sequential morphological changes occurring in sheep choroid plexus cells infected with visna virus were studied by direct immunofluorescence, acridine orange, and hematoxylin and eosin staining methods. Specific immunofluorescence was first detected in the perinuclear cytoplasm of solitary cells 24 hr after infection. As the infection progressed, viral antigen appeared in an increasing number of cells, and rounded globular cells with long slender processes harboring intense fluorescence were seen. Nuclear fluorescence was not observed in infected monolayers. Polykaryocytes formed within 6 hr after inoculation due to the direct cell-fusing effect of the virus inoculum did not show specific fluorescence. Viral antigen was found, however, in the cytoplasm of multinucleated giant cells in cover slips harvested after new infective virus had been released, and later in the course of infection circular fluorescent inclusions were seen in the cytoplasm of polykaryocytes. Comparable eosinophilic inclusions were observed in hematoxylin and eosin preparations, and acridine orange staining of infected monolayers demonstrated similar inclusions which fluoresced with the color characteristic of single-stranded nucleic acid and were susceptible to digestion with ribonuclease. Visna virus appears to be a ribonucleic acid virus which replicates in the cytoplasm.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Fixative evaluation and histologic appearance of embryonic rodent tissue

Histopathologic study of early hamster embryos was carried out after fixation in Zenker's solution, alcoholic formalin, Bouin's fluid, 10% neutral buffered formalin, or 3% glutaraldehyde and staining with hematoxylin and eosin. Fixation in Zenker's fluid followed by postfixation in neutral buffered formalin provided superior preservation of normal embryonic subcellular detail as compared to the other candidate processing techniques.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Stain Technology: Fixative Evaluation and Histologic Appearance of Embryonic Rodent Tissue

Histopathologic study of early hamster embryos was carried out after fixation in Zenker's solution, alcoholic formalin, Bouin's fluid, 10% neutral buffered formalin, or 3% glutaraldehyde and staining with hematoxylin and eosin. Fixation in Zenker's fluid followed by postfixation in neutral buffered formalin provided superior preservation of normal embryonic subcellular detail as compared to the other candidate processing techniques.     

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2‐D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2‐D clean‐up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

卵蛋白反复吸入哮喘小鼠气道重构模型的建立与评价

目的：探讨哮喘小鼠气道重构模型的建立的方法。方法：SPF级BALB／C6-8周龄雌性小鼠40只随机分成正常对照组、哮喘模型组,每组20只。哮喘组经卵蛋白（OVA）混悬液0．2ml致敏并反复雾化吸入2周、4周,正常对照组由生理盐水代替。各组分别于末次雾化激发后进行取材,收集肺组织,制作石蜡切片,HE染色观察气道中嗜酸性粒细胞;Masson三色染色法观察气道周围胶原沉积情况;PAS染色法观察气道黏液分泌情况;测定单位气道面积基底膜周径（Pbm）、管壁总面积（WAt）、内壁面积（wAi）、平滑肌面积（WAm）、胶原面积（Wcol）、粘液面积。结果：哮喘模型组WAt／Pbm、WAi／Pbm、WAm/Pbm、Wcol／Pbm、粘液分泌面积较正常对照组明显增加。哮喘4周组上述指标均高于其对应2周组（P〈0．05）。结论：反复的过敏原（OVA）吸入可导致哮喘气道重构的发生,是一种较好的建立哮喘小鼠气道重构模型的的方法。     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.