Ninhydrin Staining of Tissue Sections

This method represents a considerable improvement over earlier ninhydrin procedures. Celloidin sections were stained after mounting in a medium which clears with incubation at 55 °C. There appears to be no reason why paraffin section cannot be used. The sections were not placed in a large volume of ninhydrin (0.25% triketohydrindene hydrate in n-butanol) but only a small volume was sprayed onto the slide. Distortion resulting from heating in boiling water to develop the color was avoided by a slower treatment of 3 days' incubation at 55 °C. The use of water as a solvent in staining is also avoided, thus minimizing the possibility of color migration and insuring against the development of the intensely colored products of the ninhydrin reaction that occur in aqueous solution. Slides need not be observed upon the day of preparation, since the color was stable for about a week after its formation.     

Staining of fresh, Undecalcified, thin Bone Sections

Fresh, undecalcified bone sections can be reproducibly and reliably stained by any of the following procedures: (A) Basic fuchsin, 1% in 30% alcohol, 48 hr, 22°C. (B) AgNO₃, 0.033 M, 48 hr, 22°C; washing 48 hr in a large volume of distilled water; exposure to light to develop the color. (C) Metallic sulfides (Co⁺⁺, Pb⁺⁺, Hg⁺⁺, Cu⁺⁺): the nitrate of the metal, 0.033 M, 48 hr, 22°C; then Na₂S, 0.033 M, 48 hr, 22° C. (D) Alizarin Red S, 0.1% solution in distilled water, 48 hr, 22°C; differentiated 48 hr at 22°C in weakly alkaline water, pH about 8. (E) KMnO₄: boiling 8-10 min in a 0.1 N, solution. With the exception of D the surface stain must be ground off the section for microscopic examination of its interior. Stain concentration, time and temperature can be altered to suit specific needs.     

Electron-Microscopic Study of Osmium Ferricyanide Staining in Meristematic, Extending and Differentiated Mesophyll Cells of Winter Rye Seedlings

The osmium ferricyanide (OsFeCN) cytochemical technique wasused in the electron microscopic study of organelles in cellsof functionally different leaf zones of winter rye seedlings.In meristematic cells, the endoplasmic reticulum and nuclearenvelope were the main structures reacting with OsFeCN; in cellsof the extension zone and in differentiated mesophyll cellsit was the peripheral reticulum and the intermembrane spaceof the chloroplast envelope. The change in OsFeCN staining character of organelles betweenthe various zones of leaves is considered to be due to cellexit from the meristem. The participation of the peripheralreticulum of chloroplasts in the sequestration of Ca²⁺ ionsis proposed. Secale cereale L., leaf zones, OsFeCN-staining, Ca²⁺-sequestring organelles     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

Heavy metals may be incorporated from solution into tissue sections for electron microscopy. The resulting increase in density of the tissue provides greatly enhanced contrast with minimal distortion. Relative densities of various structures are found to depend on the heavy metal ions present and on the conditions of staining. Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood.     

Intensifier for Bodian Staining of Tissue Sections and Cell Cultures

Intensification of the standard Bodian silver stain with a poststaining photographic enhancer produces high resolution of fine cell processes such as axonal growth cones. This technique can be used on tissue sections and is especially useful for visualizing individual cells fixed in tissue cultures.     

Staining Rickettsiae in Sections of Formalin-Fixed Tissue; a 12-Minute Wright-Buffer Sequence

Rhesus monkey tissues obtained at autopsy were fixed in neutral phosphate-buffered formalin, embedded in paraffin, and sectioned 3-5 μ thick. Sections were stained with Wright's stock stain for 6 min and differentiated in Wright's stock buffer for 6 min and mounted as usual. The rickettsial cytoplasm stained reddish pink; the nucleus, blue. This method was much simpler than that of Wolbach although the results obtained were nearly identical.     

Selective Staining of Magnesium by Titan Yellow Applied to Incinerated Tissue Sections

Tissue sections were microincinerated with a Bunsen burner, allowed to cool, and coated with an 0.2% aqueous solution of titan yellow. Upon addition of 2 N NaOH, sites of magnesium deposits exhibited a flame red color, which persisted as long as the alkalinity of the mounting medium was preserved. Tests with calcium salts dissolved in serum were negative.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Improved Staining Boxes for Fast, Uniform Staining of Ultrathin Sections on Grids

A standard LKB (LKB-Produkter Ab. S161, 45 Bromma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. the staining boxes can be cut out to any required size or shape. the polymethacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant thin polymethacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. for staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. the separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

A New Method of Urinary Stone Analysis by Batik Histochemical Staining of thin Sections

Thin sections of urinary calculi are prepared by petrographic methods using Araldite of the mounting medium. By covering the remaining put of the don with wax, an exposed segment of the section is stained by a histochemical technique. By the process of dewaxing ad rewaxing, successive adjacent segments are stained by GBHA, Von Kossa, schultz, and titan yellow methods for calcium oxalate, apatite, uric acid and urates, and magnesium in magnesium ammonium phosphate, respectively. If desired, matrix in additional segments is stained with PAS and aqueous toluidine blue. Microscopic examination of each layer through all the stained segments of a stone section reveals its chemical nature. Thus the chemical composition, morphology, and spatial distribution of the crystalline and matrix constituents of thin sections of urinary calculi are simultaneously revealed in situ.     

Staining Pericellular Boutons of Glutaraldehyde-Fixed Nervous Tissue; A Chromation-Silvering Sequence for Frozen Sections

After fixing in phosphate-buffered 5% glutaraldehyde, pH 6.8, by perfusion, brains were sliced to 3-5 mm pieces which were placed in the fixative for 5-7 days. The pieces were washed through several changes of 2.26% NaH₂PO₄ for 12 hr, 30 μ frozen sections cut, and mordanted 2 days in an equal-parts mixture of 3.5% CrO₃ and 5% Na-tartrate, which had been aged at 20-25 C for 20 days prior to use. After washing in distilled water, the sections were put into a solution containing AgNO₃, 20 gm; and KNO₃, 15 gm, in distilled water, 80 ml; at 30 C for 1.5-2 hr, then reduced at 40-45 C in three pyrogallol solutions as follows: 1-2 sec in 1% pyrogallol in 55% alcohol; 3-4 sec in a 0.67% solution in 33% alcohol, and 5-7 sec in a 0.5% solution in 25% alcohol. Gold toning is optional; dehydration, clearing and covering, routine. The technic shows particularly the perisomatic fibers, boutons en passant and boutons termineaux. Fibers in nerve tracts may be visible but lightly stained; cell nuclei may be dark, but the cytoplasm remains pale.     

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.     

Selective Staining of Eosinophils and Their Immature Precursors in Tissue Sections and Autoradiographs with Congo Red

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Tissue Shrinkage Caused by Attachment of Paraffin Sections to Slides: its Effects on Staining

Sections were cut from a wide variety of tissues, and those from each block were divided into four groups before attaching and drying on slides. Four commonly accepted sources of heat were used for drying: (a) gas hotplate set at 65° C; (b) incubator, 37°; (c) oven, 56°; and (d) room temperature, 20°. After drying, the sections were stained, then examined for intensity of staining and for distortion caused by shrinkage. With both soft and decalcified tissue stained by haematoxylin and eosin, the best results occurred in the sections dried at 20° C; the next best at 37°. When stained by Van Gieson's method, both types of tissues were best after 20° drying, but the second-best group showed differences in favour of 56° for soft tissues and 37° for decalcified. After drying decalcified tissue at 65°, the staining of collagen by acid fuchsin was almost completely absent. When impregnated with silver, for reticulin, the best results for soft tissues were after 56° drying; second best, 20°; but decalcified tissues showed a reversal of this order. After PAS, there was an increasing intensity of staining from 20° to 65°, with soft tissue; evidence that histochemical interpretation could be strongly influenced by drying temperature.     

A Simple, Rapid Staining Procedure for Methacrylate Embedded Tissue Sections Using Chromotrope 2R and Methylene Blue

Sections of tissue embedded in glycol methacrylate can be stained in rapid sequence with solutions of 1% aqueous chromotrope 2R adjusted to pH 3 and 0.1% methylene blue to produce sufficient contrast and cellular detail to permit quick visual inspection and/or photomicrography. Solutions of these stains are simple to prepare and are stable over long periods. Staining of sections may be accomplished within six minutes.     

Fluorescent Staining of Juxtaglomerular Cells in Frozen Sections

Enzymatic investigations of the juxtaglomerular apparatus often creates the need for visualisation of granulated juxtaglomerular cells (JGC) in preparations subjected to histochemical procedures. In our investigations, Pitcock and Hartroft's (1958) modification of Bowie's method and the Endes et al. (1969) combined trichrome staining proved to be inadequate when applied to fresh cryostat sections, or to formol- or glutaraldehyde-fixetl, gum sucrose-impregnated frozen sections. Friedberg and Reid's (1966) crystal violet procedure for waxembedded kidneys also failed to give uniformly reproducible results. In attempting to find a satisfactory technique for both enzyme and granule staining, we noted Janigan's (1965) and Haratla's (1969) observations on paraffin-embedded JGC, and tested the following fluorochromes: thioflavine T—Fluka, C. I. 49005; auramine O—Merck, C. I. 41000; acridine orange—E. Gurr, C. I. 46005; berberine sulfate—Fluka, C. I. 75160 on 10 μ sections of albino mouse kidneys prepared in 4 different ways as follows: