Rapid Staining and Preservation of Cells Grown in Plastic Tissue Culture Flasks

Cells grown in plastic tissue culture flasks may easily be fixed and stained in large batches by first binding the flasks in groups of 6-8 and then applying all fluids with an automatic syringe, followed by decanting as required After staining, the cells are mounted in a mixture of USP mineral oil in isoamyl alcohol (2:1). In this way the cells are impregnated with a clearing and mounting agent which does not cause discoloration of the stains, does not dissolve the plastic, and is nondrying.     

In Situ Hybridization of Cytospin Preparations: A Rapid Nonisotopic Screening Method for Isolated Cells

A simple and rapid method for light microscopic in situ hybridization on cytospin preparations is described and demonstrated for detection of viral nucleic acid in a virus-infected cell line. Cells were fixed by acetone followed by chloroform, denatured by heat, hybridized at 37 C, and hybridized sites detected with a multiple step procedure (primary anti-biotin antibody, biotinylated second antibody, streptavidin-peroxidase). This method can be used for screening studies at the light microscope level, and offers a useful and simple way to determine optimum hybridization conditions for subsequent electron microscopic investigations.     

Rapid Removal of Tissue Culture Monolayers by Collodion for Subsequent Staining

Removing cultures from roller tubes before staining eliminates the destaining which often occurs when the cells are first stained and then removed by embedding in collodion. The cells are fixed in situ, dehydrated, and covered with collodion (Merk's flexible) for 10 min. The collodion is poured off, the fluid residue lining the tube allowed to dry for 10 min, and the tube is filled with tap water. The collodion cast containing the cells is loosened and removed, cut into strips, placed on slides and blotted into firm contact. The collodion is then dehydrated and dissolved with absolute alcohol followed by a 1:1 mixture of alcohol and ether. The slides can then be rehydrated and stained by conventional methods.     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

A Rapid Silver-Free Method for Staining the Myenteric Plexus

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionin were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic “mirroring,” low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.     

A Rapid Silver-on-the-Slide Method for Nervous Tissue

A progressive silver staining method is described, which permits microscopic examination of the sections during the staining process. After formaldehyde fixation, dehydration and embedding in paraffin or celloidin, fine fibers and synaptic endings may be demonstrated. After formaldehyde fixation and mordanting in 3% K₂Cr₂O₇, myelinated fibers and mitochondria are specifically stained.

The unique feature of this method is, that the silver solution (0.5% protargol) is mixed with the reducing solution: 1.6% Rochelle salts, containing traces of Ag NO₃, MgSO₄, and K₂S (U.S.P.). The sections are placed directly into this mixture, which is then warmed to 45-55° C. Sections are removed when progressive staining is completed, washed in water, dehydrated and mounted.

In the fiber stain, nerve fibers and synaptic endings are dark brown or black, and nuclear chromatin is deep brown, against a pale yellow background. When the myelin sheath procedure is followed, the fiber bundles are deep brown, and the intensity of the staining remains the same for specific tracts, aiding in their identification.     

Rapid Nuclear Staining Method for Saccharomyces cerevisiae

Mithramycin was used to stain nuclei in mitotically dividing and sporulating yeast.     

A Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however, the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl₂ · 6H₂O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Combined In Situ Zymography, Immunofluorescence, And Staining Of Iron Oxide Particles In Paraffin-embedded, Zinc-fixed Tissue Sections

AbstractSuperparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

A Rapid Screening Technique for Detection of Diosgenin Through In Situ Cytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.     

In Toto Staining and Preservation of Peripheral Nervous Tissue

A simple quantitative modification of the in toto staining technique for nervous networks of Sihler is described. The results are demonstrated on the innervation pattern of the hard palate of the rat. Formalin fixed hard palates of rat were macerated and bleached in an aqueous solution of 3% potassium hydroxide with a few drops of 3% hydrogen peroxide added. Thereafter, the specimens were decalcified in Sinter's solution I (1 part glacial acetic acid, 1 part pure glycerin and 6 parts 1% chloral hydrate), the process being controlled by radiography. The specimens were next stained in Sutler's solution II (1 part Ehrlich's hematoxylin, 1 part pure glycerin and 6 parts 1% chloral hydrate). After staining, the non-nervous tissues were destained in Sihler'g solution I. Destaining was checked microscopically and was stopped before the finest branches of the nerves began to fade. The specimens were then washed in a weak aqueous solution of lithium carbonate and cleared in increasing concentrations of glycerin. Good visualization of nervous structures and a deep field of observation resulted; orientation of the peripheral nerves with respect to surrounding structures was readily seen and a three-dimensional image of the nervous networks was obtained.     

Novel In Situ Pretreatment Method for Significantly Enhancing the Signal In MALDI-TOF MS of Formalin-Fixed Paraffin-Embedded Tissue Sections

The application of matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry (MS) to the proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue presents significant technical challenges. In situ enzymatic digestion is frequently used to unlock formalin-fixed tissues for analysis, but the results are often unsatisfactory. Here, we report a new, simplified in situ pretreatment method for preparing tissue sections for MS that involves heating with vapor containing acetonitrile in a small airtight pressurized space. The utility of the novel method is shown using FFPE tissue of human colon carcinoma. The number and intensity of MALDI peaks obtained from analysis of pretreated tissue was significantly higher than control tissue not subjected to pretreatment. A prominent peak (m/z 850) apparently specific to cancerous tissue was identified as a fragment of histone H2A in FFPE tissue pretreated using our method. This highly sensitive treatment may enable MALDI-MS analysis of archived pathological FFPE samples, thus leading to the identification of new biomarkers.     

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.     

A Method for Staining Reticulum and Red Cells in Hematopoietic Tissues of Lower Vertebrates

An aqueous solution of amido black 10B with ferric ammonium sulfate was found satisfactory for staining collagen and reticular fibrils after mordant-staining with a mixture of alizarin red S-phosphomolybdic acid, tannic acid, and toiuidine blue. This staining sequence simultaneously shows reticulum, reticular cells, and red cells in fishes, and is a new procedure useful as a routine stain for hematopoietic and lymphoid organs of lower vertebrates.     

黎豆的组织培养和快速繁殖

1　植物名称　黎豆 (Ｓｔｉｚｏｌｏｂｉｕｍｃｏｃｈｉｎｃｈｉｎｅｎｓｉｓ) ,别名猫豆、狗爪豆。2　材料类别　无菌种子苗。3　培养条件　基本培养基为ＭＳ。种子萌发及壮苗培养基 :(1 )ＭＳ Ｖｃ 0 .5ｍｇ·Ｌ-1(单位下同 ) 0 .1 %活性炭 ;诱导丛芽培养基 :(2 )ＭＳ 6 ＢＡｌ.0 ＮＡＡ 0 .1 Ｖｃ 0 .5 ;诱导愈伤组织培养基 :(3 )ＭＳ 6 ＢＡ 0 .5 ＮＡＡ 0 .0 5 ,(4)ＭＳ 2 ,4 Ｄ 0 .5 ,(5 )ＭＳ 2 ,4 Ｄ 1 .0 ,(6 )ＭＳ 2 ,4 Ｄ 2 .0 ;分化培养基 :(7)ＭＳ 6 Ａ3 .0 Ｖｃ 0 .5 ＮＡＡ 0 .0 5 ;芽体生长及诱导生根培养基…     

分蘖洋葱的组织培养与快速繁殖

1　植物名称　分蘖洋葱 (Ａｌｌｉｕｍｃｅｐａｖａｒ ｍｕｌｔｉ ｐｌｃａｎｓＢａｉｌｅｙｓｙｎ ｖａｒ ＡｇｒｏｇａｔｕｍＤｏｎ)。2　材料类别　鳞茎茎尖。3　培养条件　基本培养基为ＭＳ。愈伤组织诱导培养基 :(1 )ＭＳ ＫＴ 0 5ｍｇ·Ｌ- 1 (单位下同 ) 2 ,4 Ｄ 2 0。愈伤组织分化培养基 :(2 )ＭＳ 6 ＢＡ3 0 ＮＡＡ 1 0 ;(3 ) :ＭＳ 6 ＢＡ 0 4 ＮＡＡ 0 1。生根培养基 :(4) 1 /2ＭＳ ＩＢＡ1 5 ＮＡＡ 0 0 1。上述培养基均加蔗糖 3 % ,琼脂 0 8% ,调ｐＨ值为 5 7～5 8,1 2 1℃高温高压灭菌 1 4ｍｉｎ。培养温…