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.     

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.     

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.     

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.     

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.     

Quick Staining Method For Frozen Sections

Since the advent and general acceptance of frozen sections in histological and pathological laboratories it has been necessary to devise methods for staining these sections. The usual method is fixing the tissue to a slide by the use of celloidin. This paper is an attempt to describe a permanent, quick method of staining frozen sections without distortion or mechanical tearing of the tissues.     

Elastic Tissue Staining

It has been found that the addition of dextrin to samples of crystal violet and basic fuchsin employed in the prepararation of the elastic tissue stain after the technic of Weigert makes more sure a satisfactory final product. A modification of the original Weigert technic employing crystal violet or a mixture of crystal violet and basic fuchsin is offered as providing a better color contrast both visually as well as photographically. Crystal violet alone affords a bright greenish-yellow elastin while the addition of basic fuchsin results in a darker stain shading into dark blue as the proportion of basic fuchsin is increased.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

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.     

An Efficient Staining Method for Ultrathin Sections

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

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:     

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.     

Staining Etched Epoxy Resin Sections for Light Microscopy

Staining of etched sections for light microscopy is described. Azan staining was successful after treatment with potassium dichromate and the use of concentrated dye solutions. To remove osmium for hematoxylin-eosin staining, removal by reduction with ferrocene was used instead of oxidation. Highly selective differentiation after hematoxylin staining was achieved using p-toluenesulfonic acid-DMSO. To enhance eosin staining, a 2-bromoethylamine link between eosin and the tissue was used. Ferrocene also facilitated counterstaining of nuclei with hematoxylin after the PAS reaction. Periodic acid-methenamine silver staining was carried out without modification.     

Thin Sections from Unfixed Tissues for Histochemical Staining

Sections were cut from fresh unfixed tissues by means of a microtome provided with an apparatus for the simultaneous cooling of the knife and freezing stage. These sections were of uniform thickness and were found to be very suitable for histochemical staining. Such sections were immersed while still frozen in the fluid which contained the necessary chemicals for a specific technic. After remaining in the fluid for an appropriate time, the sections were put on slides and dried in warm air. The remaining steps were carried out on the slides. Several histochemical procedures (phosphatase, esterase, glycogen) were found to give good results when this technic was used.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.