Citrate Buffer Alternative to Picric Acid for Masson Trichrome Stain

Abstract

An alternative to picric acid as a mordant in the staining of cytoplasm, keratin, muscle fibers, and intercellular fibers in the Masson trichrome stain is a citrate acid-sodium citrate buffer used as the microwave pretreatment of the tissue mounted on a slide. The Masson trichrome stain, a combination of Biebrich scarlet-acid fuchsin and aniline blue dye can be done within 1 hr and avoids the hazards associated with picric acid, a highly explosive compound in the dry state. (The J Histotechnol 19:341–342, 1996)     

Modification of Brain Processing Techniques: Tissue Embedding in Glycol Methacrylate and Stain Modifications

Abstract

Increased interest in quantitation of the histopathological changes in a variety of neurological disorders (including neurodegenerative disease such as Alzheimer's disease) continues in an attempt to develop specific clinical-histopathological correlations. Most previous efforts at quantitation have used paraffin embedded sections of brain tissue, although plastic embedded sections have recently become a preferable alternative because they provide greatly reduced tissue shrinkage and distortion during processing, and greater clarity and improved resolution to the tissue sections. We have developed techniques for glycol methacrylate embedding and sectioning of brain tissue blocks on a standard histology laboratory microtome. In addition, we have modified routine diagnostic and investigational neurohistological stains for use in glycol methacrylate embedded brain sections, including hematoxylin and eosin, modified Bielschowsky stain, Jamarri silver technique, Einarson's Nissl stain, gallocyanin-Darrow red myelin stain, and the thioflavine-S-hematoxylin stain. The use of plastic embedded sections with appropriate stains will permit critical histopathological evaluation of nervous system tissue from patients with a variety of neurological disorders. (J Histotechnol 12:201, 1989)     

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.     

Victoria Blue-Hematoxylin and Eosin Staining: A Useful Routine Stain for Demonstration of Venous Invasion by Cancer Cells

Abstract

Victoria blue (VB)-hematoxylin and eosin (H&E) staining, originally described by Yokokawa et al in 1983, has been used extensively in our surgical pathology lab as a routine stain for all surgical specimens with cancer. This simple staining sequence is useful for the demonstration of venous invasion by cancer cells as VB dye specifically demonstrates elastic fibers in the background of H&E-stained tissues. (The J Histotechnol 13:271, 1990)     

Staining effect of Hibiscus sabdariffa extract on sperm cell morphology of Sprague–Dawley rats

Abstract

There is increasing awareness among people towards natural products. Due to their non-toxic properties, low pollution, and lower side effects, natural dyes are used in many day-to-day products. Although the African continent possesses plentiful plant resources, only a small amount has been exploited so far. This study evaluated the use of Hibiscus sabdariffa as a stain to evaluate sperm morphology. Following liquefaction, 10 μl of semen was spread onto glass slides and allowed to air-dry at room temperature. The smear was fixed for 15 minutes in methanol. The sperm morphology was analyzed by staining 10 slides of the smears with eosin (control) and the ethanolic extract of Hibiscus sabdariffa dye was used to stain the sperm cells. The smears were air-dried and viewed at magnification of ×400. Phytochemical and chromatographic analyses were carried out. The sperm cells were stained in shades of reddish brown. Preliminary phytochemical screening of Hibiscus sabdariffa revealed that it contains alkaloids, saponins, flavonoids, and tannins. Hibiscus sabdariffa has potential for use as a stain for study of sperm morphology.     

Use of Heat to Improve Connective Tissue Stains: Modifications of Masson,Movat, and Fibrin Stains

Abstract

Three connective tissue methods are presented: modifications of Masson trichrome, Movat pentachrome and a fibrin method. A modified Verhoeff hematoxylin preheated and applied in the 60°C paraffin oven was used for all methods. The Movat pentachrome modification additionally included staining with alcian blue before application of Verhoeff hematoxylin, and the fibrin method was stained with lissamine fast yellow before application of the working red stain. All sections were stained with a working dilution of Biebrich scarlet and acid fuchsin, rinsed, and differentiated with phosphotungstic acid in a 60°C paraffin oven. Demonstration of collagen in the modifications of Masson and fibrin was done with either light green or aniline blue; saffron was used in the Movat pentachrome.

All 3 techniques were improved in quality and precision with the aid of heat. Although fibrin was demonstrated in all techniques, minute quantities were better seen in the fibrin stain because the red cells were stained in a different color. These modified stains demonstrated several entities in a single slide preparation in about 20 min. (The J Histotechnol 16:349, 1993)     

Unexpected Results of Trichrome Staining of Quenched Epithelial Tissue Following Delayed Fixation

Abstract

The histological effects of freezing and thawing unfixed tissue before experimental diffusion studies across the mucosal barrier were investigated in the face of claims that such treatment was of little significance. Following fixation, tissue sections were stained by periodic acid-Schiff (PAS), alcian blue (pH 1.0 & 2.5)-PAS, Masson and Mallory trichromes, acid-picro-Mallory phosphotungstic acid-hematoxylin, Martius-scarlet-blue, luxol-fast-blue, and Sudan black B. Trichrome type staining revealed a previously unrecorded artifact, particularly evident in quenched tissues. Three distinct epithelial cell types could be identified on the basis of differential dye uptake. This finding was not evident in PAS, alcian blue, or Sudan black B stained sections. We postulate that distortion of the cellular matrix occurs as a result of the freeze, thaw, diffusion, and fix sequence and that these cells illustrate the well known phenomenon of tissue density/dye molecular size differential staining by acid dyes. (The J Histotechnol 16:343, 1993)     

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.     

Basazol Black 05L: A New Acid Resistant Metachromatic Stain for Normal and Abnormal Megakaryocytes

Abstract

Bone marrow smears fixed in formalin-95% ethyl alcohol- glacial acetic acid (FAA), stained with a paper dye known as basic black 05L, exposed briefly to 1 N hydrochloric acid, and mounted with cyanoacrylate adhesive displayed a bright and distinctive violaceous cytoplasmic color in megakaryocytes. Except for mast cell granules that stained violet, all other hematopoietic cells in normal and abnormal marrow specimens stained yellow to yellow orange. Normal and abnormal megakaryocytes, such as unusually large megakaryocytes in essential thrombocythemia and small megakaryocytes in myelodysplastic disorders, displayed this acid resistant cytoplasmic metachromasia. The stain is inexpensive, easy to use, and may prove usefu1 for identification of atypical and small megakaryocytes in myeloproliferative and myelodysplastic disorders. (The J Histotechnol 19:317–320, 1996)     

Stable Trichrome for the Demonstration of the Intermyofibrillary Network in Muscle Biopsies

Abstract

The modified Gomori trichrome, pH 3.4, has been useful in the visualization of the intermyofibrillary network, which is not obvious by connective tissue techniques of the Masson type. However, it is often capricious, requiring fresh preparation before use. The method presented, which uses three stock buffered stain solutions of the required pH, clearly demonstrates both the tissue and cell structure and has been reliably used in the diagnoses of over 600 human muscle biopsies. The solution is easily prepared and remains stable for several months. (The J Histotechnol 17:59, 1990)     

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)     

Novel Histochemical Stain for Tinctorial Detection of Cancer and Neoplastic Cells

Abstract

Zetiq has introduced a histochemical stain that claims to tinctorially identify cancer and neoplastic cells. Because of the potential importance of such a capability, we undertook to investigate Zetiq's CellDetect® staining technology as applied to cultured cell lines as well as an initial sample of clinical cases. This goal was pursued by investigating four types of comparisons: 1) cancer cell lines before and after differentiation; 2) cervical squamous-cell carcinoma (SCC) biopsies to non-neoplastic squamous epithelium; 3) SCCs to neoplastic, nonmalignant squamous epithelium; and 4) neo-plastic squamous cells to non-neoplastic squamous cells in cytological preparations. The clinical material was also stained with hematoxylin and eosin (biopsies) or Pap (cytologies) for diagnostic purposes. We found that all CellDetect®-stained cells exhibited one of the two tinctorial outcomes. Cell lines with malignant phenotype uniformly had red/purple cytoplasm, whereas the differentiated phenotype changed the color to blue/green. Moreover, these two color values are sufficiently distinct that they can be readily distinguished quantitatively with an ELISA reader when applied to cells in tissue culture. Biopsies of SCC and non-neoplastic tissues exhibit the same two color values: cancers had red/purple cytoplasm, whereas non-neoplastic tissues were green/blue stained. In contrast, neoplastic, nonmalignant tissues (dysplasias) stained red/purple, similar to SCCs. Cytological preparations gave similar results: neoplastic cells stained red/purple, whereas non-neoplastic cells exhibited green/blue cytoplasm. The study demonstrated that the staining was rapid and reproducible. Conclusion: The CellDetect® stain allows detecting cancer and neoplastic cells tinctorially based on a rapid, reproducible histochemical process.     

Binding indocyanine green to human serum albumin potentially enhances the detection of sentinel lymph nodes. An initial step for facilitating the detection of first-station nodes in penile and other urological cancers

IntroductionSurgical oncology strives to remove the primary cancer tumor together with its local lymphatic tissue. One of the techniques improving the staging of lymph nodes is sentinel node biopsy. The most common agent used in SNB is indocyanine green (ICG). Indocyanine green is characterized by its high affinity for human serum albumin (HSA). In practice, the visualization of the sentinel node is enhanced by attaching a relatively large carrier to the ICG molecule. The aim of this study was to investigate whether the covalent linking of ICG to a nanocolloid would extend the time of detection of the dye as it binds to HSA, assessed by fluorescence measurements in vitro.Material and methodsThe influence of the molar concentration of ICG on its ability to form a complex with HSA was investigated. The dye luminescence was measured, with an increasing amount of dye in the presence of a constant concentration of HSA. The stability of the ICG:HSA complex was also investigated.ResultsThe binding of ICG and human protein in a solution ratio of 3 : 1 made it possible to detect the ICG luminescence with better and prolonged visibility. In the case of the two lowest ratios, complex formation was not observed. The use of ICG bound to a nanocolloid based on human serum albumin increases the luminescence of the HSA : ICG complex up to 98%.ConclusionsProperly selected proportions of human albumin protein and ICH allowed higher and longer luminescence to be achieved. Nevertheless, further studies are necessary to establish the optimal concentration ratio.     

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)     

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).     

Helicobacter pylori stains and association between H. pylori and inflammation in gastric specimens

Gastric cancer has been strongly associated with presence of the bacterium Helicobacter pylori. To improve techniques in identifying H. pylori so that gastric cancer may be predicted early, this project was formulated to determine whether one particular stain is more effective in displaying H. pylori microscopically. In addition, this study attempted to determine whether the degree of inflammatory elements present in tissue could be used to predict the likelihood of H. pylori presence. Protocols for the staining techniques, Steiner and alcian yellow/toluidine blue (AY/TB), were employed on specimens to semi-quantitate H. pylori presence. Serial sections from the same specimens were stained with hematoxylin and eosin to determine the amount of inflammation. Spearman rho correlation was used to evaluate the association between amount of H. pylori and inflammation in each case. It was determined that AY/TB was more easily performed, more effective in demonstrating H. pylori, and more cost effective than the Steiner stain. Additionally, it was determined that a moderate positive association was indicated between high levels of inflammation and marked presence of H. pylori.     

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.     

Consistent Warthin-Starry Staining Technique

Abstract

A consistent ¹procedure for the Warthin-Starry stain technique for melanin and spirochetes is described. Close attention to solution preparation, purity, and pH, freshness of chemicals used, and rigorous adherence to the protocol is essential. The procedures and recommendations described ensure consistent success when using this often difficult stain technique. (The J Histotechnol 19:339–340, 1996)     

A Light Resistant,Permanent Eosin Counterstain For Hematoxylin

Abstract

Many eosin dyes now sold stain cytoplasm in varying shades, often masking the tissue components under study. We use an eosin Y solution as a counterstain for consistent, permanent contrast among tissue components, even in the presence of light.     

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)