The application of Miller's elastic stain to glycol methacrylate tissue sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Iron gallein elastic method---a substitute for Verhoeff's elastic tissue stain.

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration, sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections are counterstained with a variant of Van Gieson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff's elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

Iron Gallein Elastic Method—A Substitute for Verhoeff'S Elastic Tissue Stain

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration. sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections am counterstained with a variant of Van Girson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff s elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.     

A Modified Verhoeff-Van Gieson Elastin Histochemical Stain to Enable Pulmonary Arterial Hypertension Model Characterization

Optimal histochemical staining is critical to ensure excellent quality stained sections to enable light microscopic and histomorphometric image analysis. Verhoeff-van Gieson is the most widely used histochemical stain for the visualization of vascular elastic fibers. However, it is notoriously difficult to differentiate fine elastic fibers of small vasculature to enable histomorphometric image analysis, especially in organs such as the lung. A tissue fixation procedure of 10% neutral buffered formalin with subsequent fixation in 70% ethanol further compounds the problem of small vessel staining and identification. Therefore, a modified Verhoeff’s elastin stain was developed as a reliable method to optimally highlight the internal and external elastic laminae of small arteries (50-100 µm external diameter) and intra-acinar vessels (10-50 µm external diameter) in 3 µm thick lung tissue sections from models of pulmonary arterial hypertension. This modified Verhoeff’s elastin stain demonstrated well-defined staining of fine elastic fibers of pulmonary blood vessels enabling subsequent histomorphometric image analysis of vessel wall thickness in small arteries and intra-acinar vessels. In conclusion, modification of the standard Verhoeff-van Gieson histochemical stain is needed to visualize small caliber vessels’ elastic fibers especially in tissues fixed in 10% neutral buffered formalin followed by additional fixation in 70% ethanol.Key words: Histochemical stain, histomorphology, lung, Verhoeff-van Gieson, elastin     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

A rapid connective tissue stain for glycol methacrylate embedded tissue

No reliable connective tissue stains for GMA sections were available until recently. However, the use of toluidine blue in combination with basic fuchsin appeared to be a rapid and reliable connective tissue stain for glycol methacrylate (GMA) embedded tissue.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

Enhancement of Histological Detail Using Metanil Yellow as Counterstain in Periodic Acid Schiff's Hematoxylin Staining of Glycol Methacrylate Tissue Sections

Histological detail in sections from tissues embedded in glycol methacrylate was improved by counterstaining PAS/iron-hematoxylin stained sections with a dilute solution of metanil yellow. The addition of the counterstain increases contrast in tissue sections and highlights PAS-positive entities. The staining protocol provides sharp definition of tissue morphology, differentiates cell types and other tissue components and does not produce background staining.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Hematoxylin Toluidine Blue-Phloxinate Staining of Glycol Methacrylate Sections of Retina and Other Tissues

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 μm. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 μm paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.     

Science and Art in Preparing Tissues Embedded in Plastic for Light Microscopy, with Special Reference to Glycol Methacrylate, Glass Knives and Simple Stains

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

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.     

Use of tissue-free glycol methacrylate sections as semi-permeable membranes: a simple way to shorten incubation times and to improve localization in enzyme histochemistry

Placing 2-microns sections of tissue-free glycol methacrylate on top of tissue sections is a simple way of forming semipermeable membranes to enhance enzyme histochemical staining. For demonstrating alkaline phosphatase in glycol methacrylate-embedded kidney by a standard azo dye method, such membranes enabled incubation times to be reduced to 1-2 hr, with azo dye reaction product being more crisply localized as compared to sections stained without membranes. Such effects are possible because the membranes are highly permeable to small molecules (e.g., substrate and diazonium salt), slightly permeable to molecules of moderate size (e.g., the final reaction product), and impermeable to large molecules (e.g., alkaline phosphatase and other tissue biopolymers). The implications of these findings for enzyme histochemistry and for enzyme-labeled antibody staining are discussed.     

Palladium chloride as a stain for elastin at the ultrastructural level.

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.