A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification

Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

Verwendung gefriergetrockneter Kryostatschnitte für histologisehe und histochemische Untersuchungen

Zusammenfassung Die Anwendungsmöglichkeiten verschiedener histologischer und histochemischer Techniken an gefriergetrockneten Kryostatschnitten wird beschrieben. Es wird gezeigt, wie die Schnitte auf Objektträgern montiert und in wässrige Medien eingebracht werden können. Dabei treten nach kontrollierter Gefriertrocknung weit weniger Artefakte auf als bei Weiterverarbeitung von Paraffinschnitten gefriergetrockneten Gewebes; auf eine Rehydrierung in der feuchten Kammer kann im Gegensatz zur Verwendung von Paraffinschnitten gefriergetrockneten Gewebes verzichtet werden. — Für histologisehe Untersuchungen und Mucopolysaccharid-Nachweise gibt das Aufziehen der Schnitte in reinem Methanol nach vorheriger Bedampfung mit Formaldehyd (60 min, 20° C) die besten Ergebnisse. Für Enzymnachweise ist die Fixierung in Isopropylalkohol, für Dehydrogenasen in Aceton, am geeignetsten. Dabei gelingen der histochemische Nachweis der Cholinesterasen und der lysosomalen Enzyme besser als am konventionell behandelten Kryostatschnitt.

---

The application of histological and histochemical techniques to freeze-dried cryostat sections

Summary The use of freeze-dried cryostat sections for various histological and histochemical techniques is described. It is shown, how sections can be mounted on slides and how they can be transferred into water-containing media. Following controlled freeze-drying artefacts due to watering are highly reduced as compared to paraffin sections of freeze-dried tissue; a re-hydration in a moist chamber is dispensable. — For histological purposes and investigations on mucopolysaccharides a formaldehyde vapour fixation (60 min, 20° C) followed by infiltration of the sections with pure methanol gives the best results. For enzyme histochemistry the postfixation with isopropanol is well suited, for dehydrogenase reactions acetone is recommended. — Histochemical reaction for cholinesterases and lysosomal enzymes on freeze-dried sections are superior to conventional techniques.

     

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:     

The effects of various fixatives on subsequent lectin binding to tissue sections

Summary The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin—saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate—paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin—saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.     

HISTOSCAN: Computer program for cytophotometry in tissue sections and its application in the evaluation of nuclear atypia

Summary The grading of nuclear atypia has a great and well recognized value when predicting the malignancy of neoplastic processes. Although the basic criteria for the grading are generally agreed upon, in the individual case, the final judgement is to some extent still a matter of subjectivity, which naturally impairs reproducibility. The present paper describes a method for quantification of variability of nuclear size and Feulgen-stainability. By plotting the mean optical density against the area value in a scatterdiagram, a cluster is obtained, the size of which reflects the degree of nuclear atypia. The measurements are performed in tissue sections using stage scanning cytophotometry. A computer program — HISTOSCAN — has been developed which enables measurements in highly cellular tissues. The system is also insensitive to the influence of light scattering, a factor of importance when measuring in tissue sections. The performance of the program is tested in both imprints and tissue sections.This work was supported by the Swedish Cancer Society, grant no. 1284-B80-02X     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

The correlation between uptake of Methyl Green and Feulgen staining intensity of cell nuclei. An image analysis study

Summary Paraffin sections of rat tissue fixed in either formaldehyde solution (3.6% w/v) or in Carnoy's fluid were stained using standardized Methyl Green—Pyronin procedures with the dyes used either simultaneously or in sequence. The sections were evaluated for the uptake of the two dyes by cell nuclei, nucleoli and cytoplasm using colour TV-image analysis. The parameters measured were integrated optical density and the surface area of the object. The sections were then destained and a Feulgen reaction was performed. The coordinates of the cells measured after the simultaneous Methyl Green—Pyronin method were stored in the computer, making it possible to measure the same cells in the Feulgen-restained sections. Image analysis gave results which invalidate the sequential methods as opposed to the simultaneous method. Mean optical densities were significantly increased for both dyes with the simultaneous method after formaldehyde fixation as compared to Carnoy fixation. The quantitative correlation of Methyl Green and DNA in the simultaneous technique was found to parallel exactly that of the Feulgen stain. In conclusion, the simultaneous Methyl Green—Pyronin technique is recommended while the sequential methods seem to be of less value.     

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections

Summary A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen— and antibody—enzyme conjugates and their application in single, double or triple staining studies are reviewed.The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen—antibody complexes.     

Enzyme histochemistry and immunohistochemistry with freeze-dried or freeze-substituted resin-embedded tissue

Summary Freeze-drying or freeze-substitution, combined with low-temperature resin-embedding, represents a new approach to the optimum preservation of tissue for enzyme histochemistry and immunohistochemistry. This method, which avoids tissue fixation, combines excellent tissue morphology with the preservation of enzyme activity and immunoreactivity and allows high-resolution enzyme histochemical and immunohistochemical studies to be performed. The activity of a wide range of enzymes can be demonstrated in sections of freeze-dried or freeze-substituted resin-embedded tissue. Enzymes are retainedin situ with high activity, accurate localization and no diffusion. Immunohistochemical studies can also be performed on resin sections, and antigens—especially labile antigens — are immobilizedin situ without denaturation and can be demonstrated with high sensitivity and accurately localized. This method allows the localization and distribution of enzymes and antigens to be studied in relation to excellent histological and cytological detail.     

Bulk Handling Racks for Free-Floating Serial Sections, Especially Suitable for Nauta Staining

Staining racks, each containing 20 shallow compartments, were constructed by drilling 1.5 cm holes in 8 × 11 cm sheets of 1 mm thick Darvic, an unplasticised polyvinyl-chloride compound, and cementing fibre-glass gauze of 1.3 mm mesh size to one surface. Sections were placed serially—one to each compartment—in the racks, and stacks of up to 9 racks were clamped by Perspex (methyl methacrylate) nuts and bolts, and side clamps. Thus, sections could be handled easily and kept in strict serial order, even in bulk. For Nauta staining, brains had to be gelatin embedded before sectioning. By storing sections in groups of 4 in ice-cube trays, evenly spaced series could be selected for placement in the racks. As many as 160 sections were taken together through all stages of the Nauta method, the timing of critical stages being controlled by taking 4 to 6 free-floating sections, together with the racks, through the various solutions.     

Histoautoradiographic Localization of Calcium in Oat Plant Tissues

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Improved avidin-biotin-peroxidase complex (ABC) staining

Summary A considerable intensification of the avidin—biotin—peroxidase complex staining system (ABC) was obtained by sequentially overlaying the sections to be immunostained with an avidin-rich and a biotin-rich complex. Each sequential addition contributed to the deposition of horseradish peroxidase on the immunostained site and allowed the subsequent binding of a complementary complex. With this technique a higher dilution of the antisera could be used and minute amounts of antigen masked by the fixative could be demonstrated on paraffin sections.     

Selective Staining of Protein and Starch in Wheat Flour and its Products

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H₂SO₄ per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.     

Accurate Placement of Ultrathin Sections on Grids; Control by Sol-Gel Phases of a Gelatin Flotation Fluid

A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.     

The demonstration of anions generated by the action of sodium hypochlorite (NaOCl) on tissue sections,including observations on the unmasking of carbonyl groups

Summary Treatment of 4 micron-thick plastic sections of animal tissues with dilute solutions of NaOCl resulted in increased basophilia of tissues. A number of staining mixtures which selectively demonstrated the NaOCl-generated acid groups were then developed. Two distinct acid groups — sulfates and an unknown anion — were found to be generated by NaOCl. It was also determined that treatment of sections with NaOCl resulted in the unmasking of carbonyl groups associated with elastin and other structures.This work was done under Public Health Service Contract No. 5 Tol ES 00038-02.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.