Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

Silver Staining of Ultrathin Sections of Tissues Embedded in Polyester Resin

Specimens 1 mm³ from rat liver and kidney were fixed for 50 min in cold (0-2° C) 1% OsO₄ in veronal-acetate buffer, pH 7.7, and containing 0.1% MgCl₂; then dehydrated and embedded in Vestopal-W. Sections were cut in two ranges, 0.1-2 µ and 60-90 mµ thick, and attached to slides by floating on water and drying at 60° C. The thicker ones, for light microscopy, were soaked in acetone 1.5-3 hr; the thinner, for electron microscopy, 20-30 min. Both kinds were stained by Wilder's (1935) method for reticulum. Those for light microscopy were finished by dehydrating, clearing and covering in the customary manner; those for electron microscopy, by coating with 1% parlodion, drying, cutting the film about 2 mm² around the section, and freeing the section by soaking in water. The section was then mounted on a grid. The structures stained are: nuclei, basement membrane of capillaries, reticulum fibers of the liver and kidney, and in addition, the basement membrane of the kidney tubules. The mitochondria, vesicles, endoplasmic reticulum and cell membranes were not defined.     

The Staining Of Acid Fast Bacilli In Sections Of Glycol Methacrylate Embedded Tissues

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

Pyronin Y For Staining Stripped Autoradiographs Prepared from Plastic Embedded Sections of Rat Tissues Containing Plutonium

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.     

Differential Staining of Calcified Tissues in Plastic Embedded Microtome Sections by A Modification of Movat's Pentachrome Stain

Movat's pentachrome I stain has been adapted and modified as a stain for Undecalcified bone sections. After embedding in methyl methacrylate, this procedure yields consistently good results, with an excellent and colorful contrast between mineralized and unmineralized compartments of both cartilage and bone. in addition, osteoblats, osteoclasts, and other cells and tissue components can easily be differentiated. the staining properties of the lacunar wall surrounding the osteocytes are considered to reflect various states of osteocytic activity. the method is especially useful for the study of bone growth and bone repair, and as a stain for conventional histomorphometry and computer-assisted image analysis in bone biopsies.     

Manipulation of Tissues for Improved Aceto-Carmine Staining

The importance of the study of the structural-mechanic properties of any particular material in order to improve the technic for aceto-carmine smears and to obtain better preparations of that material has not been, perhaps, sufficiently emphasized in the large number of papers on such cytological technic. The usefulness of such a study will be shown here in two cases met by the writer.     

Cytological Staining Procedures Applicable to Methacrylate-Embedded Tissues

Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast microscope, mitochondrial preparations become routinely available without the uncertainties of most of the mitochondrial staining methods. It appears, therefore, that plastic embedding should find a useful place among the methods for light microscopy as well as in the armamentarium of the electron microscopist.     

Staining Cell Constituents in Diseased Plant Tissues

Tissues of diseased plants, where embryonic cells, with dense cytoplasm, are to be studied cytologically in the same section with vacuolated cells, should best be killed with the Meves or Regaud fluids. The greatest changes in affected cells of diseased tissues are likely to occur in the vacuolar system, the contents of which should be well preserved in the killing process.

Staining with acid fuchsin and decolorizing with light green, makes it possible, in properly killed tissues, to detect the slightest alteration in tissues, and to observe the different parts of the cell, nucleus, mitochondria and plastids, even when excessive vacuolation compresses the cytoplasmic inclusions.     

Staining Paraffin-Embedded Plant Tissues with Acridine Orange

Stem, leaf, and bud tissue of sweet potato, tomato, and pepper were embedded in paraffin, sectioned, mounted, and stained with 0.01, 0.1 and 1% aqueous and 0.1% alcoholic solutions of acridine orange. Temporary and durable mounts were prepared and irradiated under short and long wave ultraviolet light. Intensity and specificity of the fluorescence imparted to tissues were chiefly affected by type of fixative. Best results were obtained with fixatives containing formalin but not acetic acid. Tests on the effect of pH obtained with McIlvaine's buffer between 4.5 and 8.3, and made only with the aqueous stain, showed 6-8 to be optimal. Aqueous staining 1 hr in 0.1% solution, pH 6-8 is recommended for temporary mounts. Durable mounts in a nonfluorescent resin can be made after differentiation in buffer and dehydration in dioxan solutions.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

Staining Scab Actinomyces in Potato Tuber Tissues

Actinomyces hyphae imbedded in the middle lamellae of potato tuber cells may be stained in sections by the use of a modified Gram's stain. The modifications are: a very strong (5%) solution of crystal violet in anilin oil; a 24-hour exposure to both the dye and the iodine solution; and a slow decolorization in absolute alcohol until no more color flows.     

An Adaptable Staining Schedule for Plant Tissues

A general schedule for staining meristematic, maturing, and mature plant tissues is described. Treatment with a dilute aqueous solution of Delafield's hematoxylin is followed with staining in 0.1% safranin in 60% alcohol. Destaining of safranin may be partly accomplished in alcohol and completed by counterstaining with dilute fast green FCF in a xylene and alcohol mixture. Various modifications and adaptations are briefly discussed.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

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.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

A Staining Combination for Phloem and Contiguous Tissues

A technic is described for producing critically stained preparations of phloem tissue. The preparations promise to be relatively stable. Sections of fixed unembedded or of embedded (paraffin or celloidin) phloem, cambium, and xylem are (1) stained in Foster's tannic acid-ferric chloride combination; (2) treated with 1% NaHCOg in 25% or 50% ethyl alcohol for 30 minutes; (3) stained in a saturated solution of lacmoid (made alkaline by adding a few ml. of 1% NaHCO₃ in 25% alcohol) for 12 to 18 hours; (4) dehydrated and cleared in a series composed of 1% solution of NaHCO_s in 50% ethyl alcohol, 80%, 95%, and absolute alcohol, equal proportions of absolute alcohol, clove oil, and xylene, and finally pure xylene; and (5) mounted in a neutral resin. Callose and lignified secondary walls are blue or blue-green in color, cellulose walls and stainable protoplasmic contents are generally light brown. The technic has been successful with sections from 5 to 40μ in thickness, and the staining has been satisfactory for both color and black and white photomicrography.     

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.     

Fixing and Staining of Conifer Tissues with Lacto-Propiono-Dimethylsulfoxide Carmine

During a study of the development of callus from immature microsporophylls and megagametophytes of Pinus spp. (Bonga 1974), tissue squashes were stained with aceto-carmine (Gerlach 1969). However, this stain had some drawbacks. The cells and nuclei formed in the pollen and pollen tubes during the early stages of culture were often obscured by large numbers of granules (cf. Christiansen 1973), and often, small groups of small vigorous cells within the callus failed to stain.