Modification of the silver impregnation technique of Bielschowsky for use in glycol methacrylate-embedded brain tissue

The modified Bielschowsky stain has become the standard silver impregnation technique for use in paraffin-embedded tissue sections for identifying abnormalities of neuritic processes in a variety of neurodegenerative disorders such as Alzheimer's disease. Recently, glycol methacrylate embedding has been used increasingly as a substitute for paraffin embedding of tissue, because it results in less tissue distortion during processing and provides greater clarity and resolution of cellular architecture and structural details. We have been able to modify the Bielschowsky stain for use in glycol methacrylate-embedded brain sections. The use of this technique will permit more accurate quantitation of the histopathologic changes in neuritic processes in neurodegenerative diseases.     

A Toluidine Blue Method for Demonstrating Mast Cells

Abstract

A modified Giemsa azure-eosin method for staining polymethyl methacrylate (PMMA) plastic embedded bone sections is described. The method is similar to those used to stain glycol methacrylate sections but differs in timing and addition of air drying steps. The stain yields effective differentiation of marrow cells, growth plate, calcified cartilage, osteoid, bone matrix and bone cells in undecalcified sections of bone. The stain does not interfere with the resolution of fluorochrome labeled specimens. Hence, the same slide can beused to evaluate static and dynamic parameters of bone metabolism. (The J Histotechnol 14:85, 1991)     

Volume 13, March 1990—December 1990

Abstract

A microwave Giemsa method for paraffin embedded tissue sections is reported. It is a modification of an earlier stain by Churukian and Listinsky, in which warm glycerol is used to dissolve the dyes and Triton X-100 is added to the final solution. The procedure can be completed within one hour and fifteen minutes and is consistent and reliable for demonstrating a variety of cells and microorganisms, like rickettsia and gram-negative bacteria. The stain can be easily prepared in the laboratory, does not require a microscope to control differentiation, and is as good, if not better, than commercially available Giemsa stains. Results are more uniform and consistent than those obtained with the widely used Wolbach method. (The J Histotechnol 18:319, 1995).     

Dibasic staining of large epoxy tissue sections and applications to surgical pathology

A variety of normal and pathologic aldehyde-fixed osmium postfixed human tissues were prepared as large sections embedded in Spurr epoxy. They were stained with a sequential basic fuchsin--methylene blue stain which gives "hematoxylin- and eosin-like" staining and additionally functions as several special stains. This technic also allows for electron microscopy directly on the embedded tissue. The histologic and cytologic preservation and overall staining was superior to tissue embedded in glycol methacrylate. The methods and technics presented in this article have important applications in diagnostic surgical pathology and histology in general.     

Silver Labeling of Alzheimer Neurofibrillary Changes and Brain β-Amyloid

Abstract

Alzheimer neurofibrillary changes and brain β-amyloid can be stained selectively by silver impregnation on tissue sections and (in the case of neurofibrillary proteins) on sodium dodecyl sulfate-polyacrylamide gels. The stain can be applied to frozen sections of tissue from 10–150 μm thick, to similarly thick polyethylene glycol sections, or to 5–15 μm thick paraffin sections. The technique can also be applied to routinely fixed autopsy material. The technique takes advantage of the physical development of nucleation sites, thereby permitting tight control of the entire procedure. It is less expensive than immunocytochemical techniques, and facilitates processing of large numbers of sections through entire hemispheres of the human brain. (The J Histotechnol 16:335, 1993)     

A Rapid Stain for Campylobacter pylori in Gastrointestinal Tissue Sections Using Diff-Quik®

Abstract

Campylobactw pylori is a spiral bacteriurm recently recognized as a highly prevalent organism in the upper gastrointestinal tract. Because of its suspected pathogenetic role in gastritis and related disorders, histologic identification of the organism is often desirable. We describe a simple, rapid method for demonsmting the organism in paraffin sections of gatrointestinal tissue utilizing a commercially available, premixed min (Diff-Quick®) that gives results similar to more cumbersome Giemsa-type tissue stains. (The J Histotechnol 12:303, 1989)     

Extraction and Re-embedding Technique for Salvaging Impaired Glycol Methacrylate Specimens

Abstract

This paper describes the detailed salvage technique used for research specimens that had been infiltrated and embedded in glycol methacrylate (GMA). During the second infiltration step the GMA spontaneously polymerized, “trapping” the tissue cassettes in a “mass” of prematurely polymerized plastic. After the tissue was successfully “reclaimed,” the cassettes were reinfiltrated with catalyzed GMA, embedded, cut and successfully stained. (The J Histotechnol 12:53, 1989).     

Staining efficacy assessment of a differential routine and special stains for pathological stromal calcifications in maxillofacial lesions

ABSTRACT

Head and neck connective tissue lesions may have diverse calcifications within the fibrous connective tissue stroma. The perplexity involved in the identification and determination of the nature or degree of calcification through routine hematoxylin and eosin (H&E) stains necessitates the usage of a specific, simple, and cost- and time-effective differential staining techniques. The aim of the present study was to develop criteria to distinguish bone formation from bone resorption using methylene blue-acid fuchsin (MB/AF) stain and the role of collagen fibers in the identification of stromal calcifications using polarizing microscopy with picrosirius red stain. Twenty cases with pathological diagnoses for various stromal calcifications in maxillofacial lesions were retrieved from the departmental archives. Decalcified formalin fixed paraffin embedded tissue sections were stained with hematoxylin and eosin, Masson’s trichrome (MT), methylene blue-acid fuchsin (MB/AF), and picrosirius red. The stained sections were assessed to identify the calcifications found in the surrounding connective tissue stroma. It was observed that most cases showed maximum staining intensity with MB/AF stain as compared to the other staining methods. Moreover, the results suggested that contrast between calcification and stromal soft tissue was best distinguished with the MB/AF stain except in the case of dystrophic calcifications. Along with this, polarizing microscopy with picrosirius red enables better characterization of stromal components. Although the H&E stain and a connective tissue stain i.e. Masson’s trichrome, are employed routinely in histopathology; the use of special stains such as MB-AF and picrosirius red facilitates the identification of calcifications from the stromal tissues.     

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Abstract

Toluidine blue stain has been widely used for staining undecalcified bone sections, but previous techniques required removal of embedding plastic or surface etching with both alcohol and acid solutions. In this study, solutions of toluidine blue at different pHs were used to stain unetched and acidetched sections. A group of the test staining solutions were heated in an effort to enhance section staining. Acid-etching prior to staining was found to be necessary to achieve bone matrix staining, but methanol etching could be eliminated if staining solutions were heated to 55–60°C. Sections stained with the heated solutions buffered to pH 7.0 or 8.0 had optimum morphology. (The J Histotechnol 17:357, 1994)     

Subject Index

Abstract

Acrylic resin mixtures are now widely used as embedding media for the preparation of tissue sections. Most of these mixtures are based on 2-hydroxyethyl methacrylate (glycol methacrylate, GMA). Resin embedding preserves tissue components far better than paraffin, celloidin or frozen sections. The present review describes the basic principles and trouble shooting, in particular: the chemical and physical properties of GMA, and components used for GMA mixtures; fixation of tissues for resin embedding; methods for dehydration; microtomy; stretching on water and mounting in relation to the final dimensions of GMA sections; staining of GMA 3embedded tissue sections; and the use of GMA resins in immunohistochemistry. In addition, standard, step by step procedures for embedding tissues in GMA is included. (The J Histotechnol 19:297–311, 1996)     

Comparison of five elastin histochemical stains to identify pulmonary small vasculature*

Optimization of an elastic tissue histochemical stain to enable clear, crisp visualization and quantification of pulmonary small vasculature is central to the histomorphologic quantitation of pulmonary vasculature wall thickness. To accomplish elastic tissue histochemical stain optimization, five histochemical elastin stains were compared to identify the internal and external elastic laminae of small arteries (50–100 μm in external diameter) to very small intra-acinar vessels (10–50 μm in external diameter) in rat lung tissue sections. The five elastin stains included: a modified Verhoeff’s elastin stain, Miller’s elastic van Gieson, and three modifications of the Miller’s stain. The Miller elastin stain is a progressive procedure that does not require a differentiation step, thus enabling consistency and reliability of staining from slide to slide. A modified Miller’s histochemical staining methodology successfully highlighted the pulmonary small caliber vasculature wall thickness. The modified method was technically easier and less time consuming to perform than regressive methods. To improve elastin-to-background contrast, modifications to the Miller’s stain included bypassing the nuclear staining and using a neutral red counterstain in place of the van Gieson counterstain, both of which greatly facilitated observer-assisted pulmonary vascular structure identification for histomorphometric quantitation.     

A Tinctorially Distinctive Hematoxylin and Eosin Stain with Relative Simplicity and Consistency

Abstract

Paraffin embedded sections fixed in 10% formalin were stained in Shandon Instant Hematoxylin, differentiated in acid alcohol, blued in dilute sodium acetate, and counterstained with a concentrated eosin-multichrome stain. This simple procedure produced consistent results with deep blue nuclei and bright counterstaining. (J Histotechnol 12:210, 1989).     

Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Abstract

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)     

A Polychrome Stain for Mucopolysaccharides on Semithin Sections of Epoxy Resin Embedded Tissues

Abstract

The method describes a simple, quick polychromatic stain for Epon-Araldite and Araldite embedded tissue. It differentially stains mucins and mucopolysaccharides. The technique is simple to use and requires a hot plate to carry out the stain, which is done with droplets of Milipore filtered stain solution. It is useful as a screening stain or as a stain for some tissue components that generally require complex and prolonged procedures. A wide range of shades are produced, with the best effects achieved in tissues with abundant connective tissue, mucopolysaccharides, or granulocytic cells. Intraluminal bacteria of gastric biopsies are also demonstrated.     

Glycol Methacrylate for Routine,Special Stains,Histochemistry, Enzyme Histochemistry and Immunohistochemistry: A Simplified Cold Method for Surgical Biopsy Tissue

Abstract

A method of preparing surgical pathology bone marrow, lymph node, and kidney biopsies for glycol methacrylate embedding, while preserving enzymes and antigenicity in each tissue biopsy block, is described. Biopsy tissues are fixed in cold phosphate buffered formalin at pH 7.3, rinsed in cold phosphate buffer, dehydrated, infiltrated and embedded in cold glycol methacrylate, then polymerized at 4°C. Sections are cut at 1–2 pm in thickness with disposable blades or glass knives. Room temperature drying of sections and slides preserves enzymes. Antigenicity is preserved when drying slides at room temperature but, due to section removal during fluorescence staining, it is necessary to incubate slides at 37°C. This technic has proved to be a valuable tool in diagnostic biopsy work.     

A Rapid Method For Methyl Methacrylate Embedding of Mineralized Bone Specimens

Abstract

A method for methyl methacrylate embedding of mineralized bone specimens is described which takes only one week to complete. The resulting tissue block is easy to section and the four micron thick sections can be deplasticized and stained with routine or special stains.     

Histotechnologists,the Color Index and dye purity

Abstract

Histotechnologists skillfully use histochemical stains to demonstrate various components in tissue sections with different colors, and most histology laboratories have available a wide range of biological dyes to stain histological tissue sections. Histotechnologists can improve their skills by paying attention to the Color Index number for each dye they use, i.e. a special five digit number assigned to each dye to separate it from any other dye used in the histology laboratory. This short article reviews briefly the use of the Color Index, dye generic names and dye purity.     

Plastic-embedded human marrow biopsy specimens: improved histochemical methods

Improved methods for processing, sectioning, and staining plastic (glycol methacrylate)-embedded human marrow biopsy specimens were studied. Special stains, including naphthol AS-D-chloro-acetate esterase, PAS, reticulin, and iron, have been modified so that they are suitable for undecalcified, 2-microns-thick, plastic-embedded human marrow biopsy specimens. These adaptations permit plastic-embedded marrow specimens to be used for clinical diagnosis. Marrow biopsy specimens embedded in plastic were compared with biopsy specimens preserved by the conventional paraffin method. The plastic-embedded marrows provide better results from morphologic examination (enhancing diagnostic accuracy), permit assessment of bone as well as of marrow, and allow histochemical analysis to be performed.     

The Denn-Golgi stain — A Modified Defano's Silver Impregnation Stain for Dendritic Spines and Neurofibrils

Abstract

A modification of Defano's method for Golgi apparatus is described with emphasis on reduced overall stain time and better stain definition. This method utilizes a variation of widely used silver nitrate impregnation solution modified for a specific research need. The addition of a counterstain provides improved contrast and definition of dendritic spines in the Golgi apparatus of brain tissue. This modified method provides consistent, reproducible results for large numbers of brain specimens, and as such is believed to be superior to the Defano method.     

Variation of the Holmes Method for Histologic Staining of Bone Canaliculi

Abstract

The Holmes method for soft tissue staining of nerve fibers was modified to produce excellent detail of the canalicular network in decalcified bone tissue. With simple changes in timing and the meticulous use of fresh reagents, we were able to stain bone lacunae and canaliculi in a variety of animal and human tissues. This technique produced more uniform and accurate results than the thionin/picric acid stains used previously for visualization of osteocytes. The morphologic results can be used for diagnostic purposes as well as stereological studies where the osteocyte network is of importance. (The J Histotechnol 16:355, 1993)