Staining Processed Radioautographs

Integrated radioautographs obtained by coating with melted nuclear emulsion (Eastman's NTB3) are serially processed and stained at 4° C. Kingsley's mixture of methylene blue-azure A at pH 6.9 is followed by 0.05% NaHSO₃, which destains the emulsion. Basic fuchsin, 0.05% aqueous, is used as counterstain, followed by dehydration in absolute ethanol, clearing in cedarwood oil and mounting with Canada balsam. Basic fuchsin alone produces an oftentimes satisfactory monochromatic background.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 70 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 60 mA/cm² and 34 3C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 7.0 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 +/- 60 mA/cm2 and 34 degrees +/- 3 degrees C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

Method of counterstaining preparations for immunofluorescent studies of the pituitary]

To elucidate nonfluorescent structural elements of the hypophyseal parenchyma for immunofluorescent investigations, properties of some dyes most commonly applied for hypophysis staining have been studied. Such dyes as paraldehide-fuchsin, light green, orange G, chromotrop 2R, hematoxylin, eosin, fuchsin, azocarmin possess their own intensive luminescence and block immunofluorescence completely. Some other dyes (trypan blue, bromthymol blue, aniline blue, malachite green, methyl green) though not blocking immunofluorescence, they do not reveal hypophyseal cellular elements distinctly enough. Good results have been obtained with 0.3% water solution of toluidine blue, 0.5% solution of methylene light blue, methylene blue, as well as with Gram--Weigert's staining and with gallocyanin after Einarson. For special staining of corticotropocytes, the authors recommend 0.1% solution of bromphenol blue in barate buffer, pH 8.2.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S.

A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue.     

Rapid chemical dehydration of biologic samples for scanning electron microscopy using 2,2-dimethoxypropane.

Acidified 2,2-dimethoxypropane (DMP) has been used as a rapid chemical dehydrating agent en route to plastic embedding and ulthrathin sectioning for conventional electron miscroscopy (J Histochem Cytochem 23:107, 1975). We have used DMP to dehydrate biologic specimens prior to critical point drying and metal coating for scanning electron microscopy. There is no difference in either the gross architecture or the fine surface structure of mouse small intestine and trachea, rat trachea and kidney, and cultured fibroblasts, between samples dehydrated in DMP for 5 min to 30 days and those conventionally dehydrated in ethanol or acetone. DMP dehydration is advantageous in speed, economy and apparent completeness.     

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.     

Localization of methylene blue paramolybdate in vitally stained nerves

Methylene blue taken up by living neurons can be preserved for electron microscopy in a fixative containing osmium tetroxide and ammonium paramolybdate at pH 5.2. Paramolybdate is the buffer, precipitating agent and main osmotic ingredient; it does not function as an electron stain unless methylene blue is present. The low pH keeps the dye/paramolybdate complex from dissolving. Neither the low pH nor drastic dehydration from water to absolute ethanol harm the tissue. The staining mechanism involves cationic methylene blue associating with anionic structures such as microtubules and neurofilaments in the living cell; during fixation paramolybdate forms a precipitate with the dye at the staining sites. This fixative does not preserve microtubules unless they are first vitally stained.     

Staining Glycol Methacrylate Embedded Cartilage with Triethyl-Carbocyanin Dbtc (“Ethyl-Stains All”) With Special Reference to the Interlacunar Network

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.     

Preservation of fine structure in vibratome-cut sections of the central nervous system stained for light microscopy

A technique without negative effects on tissue preservation that allows precise identification and subsequent removal of central nervous system nuclei for ultrastructural analysis is described. The procedure uses 200 microns thick Vibratome-cut sections of glutaraldehyde fixed brains. These sections are stained for 25 seconds with a methylene blue solution and stored for 4 hours in 0.2 M pH 7.4 phosphate buffer in 4% sucrose for optimal visualization at the light microscopic level. The stock solution of 1 g methylene blue and 1 g sodium borate in 100 ml of distilled water, is filtered through a Millipore filter and diluted 5:95 with distilled water immediately prior to use. Regions of specific interest are then processed for electron microscopy.     

An interesting case of colloidal iron positivity

Summary An interesting case of a colloidal iron (CI) positive basophilic substance in the adrenal medullary cells of amphibia and reptilia is reported here. The substance, however, does not stain by alcian blue (AB). It is negative to PAS, Azure A, aldehyde fuchsin, AB at pH 1 and MgCl₂ — AB though orthochromatically stained by toluidine blue at pH 3. More work is needed to establish the exact nature of the CI positive material.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

Stains Compatible with Dipping Radioautography

Paraffin sections of 13 different kinds of mouse tissues, containing tritiated thymidine, were used to test stains applied either before or after application of the nuclear emulsion by dipping. Criteria used to determine compatibility were good histological definition with moderate gelatin coloration, sharp contrast to silver granules and no artifact or bleaching. Stains which worked best when applied prior to dipping included Feulgen-fast green, chronium hematoxylin-phloxine, and aldehyde fuchsin-PAS. Stains which worked best when performed after photographic development included celestin blue-Mayer's haemalum, metanil yellow-iron hematoxylin, lithium carmine-picric acid, Weigert acid-iron hematoxylin, alum cochineal, methyl green-pyronin, indigo carmine-picric acid, methylene blue-azure A, toluidine blue, Nissl and Cason. “Combination” stains which worked well when tissues were partially stained before dipping and completed after development included trichrome-PAS, luxol fast blue-PAS, hematoxylin-eosin, and aniline blue-carbol fuchsin.     

A rapid connective tissue stain for glycol methacrylate embedded tissue

No reliable connective tissue stains for GMA sections were available until recently. However, the use of toluidine blue in combination with basic fuchsin appeared to be a rapid and reliable connective tissue stain for glycol methacrylate (GMA) embedded tissue.     

Identification of Basic Nuclear Proteins by their Boronate Complex

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.     

Identification of Basic Nuclear Proteins by their Boronate Complex

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.     

A Staining Sequence for A, B and D Cells of Pancreatic Islets

Recent investigations strongly suggest the elaboration of a third pancreatic hormone by the D cell and the existence of cells which show the staining properties of both B and D cells. Demonstration of these and all other islet cells in a single section is possible by the following staining sequence: (1) of D cells by silver or toluidine blue, (2) of B cells by pseudoisocyanin, and (3) empirical staining of all islet cells together by aldehyde fuchsin, ponceau de xylidine, acid fuchsin and light green. Difficulties in embedding compact pancreatic tissue can be overcome by dehydrating to 80% ethanol, followed by tetrahydrofurane as the intermediate fluid to paraffin infiltration.     

Gross Differential Staining of the Hypophysis

The lobes of the hypophysis of many mammals can be differentiated by staining either the entire gland for 5–8 hr, or gross 1–2 mm slices of the gland for 3–5 min in a mixture of acid fuchsin (National Aniline, certified Andrade indicator) and methylene blue (Fisher certified reagent). For best results, the staining mixture contains 0.8–1.0% acid fuchsin and 0.2–0.4% methylene blue, both made up in 10% formalin adjusted to pH 6.70–6.75 with phosphate buffer. The anterior lobe stains a light blue, the intermediate lobe a dark blue, the posterior lobe a light pink and the capsule a dark pink.     

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H⁺ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.