A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Replacing xylene with n-heptane for paraffin embedding

Abstract In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

Microwave Fixation of Whole Fetal Specimens

This study compares microwave fixation of whole fetal specimens with conventional techniques performed at room temperature. All fetuses were obtained from the same pregnant rat; half of them were placed in neutral formalin for 15 min at room temperature, then irradiated for 2.5 min in a domestic microwave oven. The remaining fetuses were placed in neutral formalin at room temperature for 48 hr as a control. Both experimental and control groups were exposed to routine tissue processing for light microscopy and embedded in paraffin wax. Sections 5 μm thick were stained with hematoxylin and eosin. Our results showed that the microwave technique reduced the fixation time while providing thin sections that were equal to or better in quality than those in the control group.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

Deparaffination time: a crucial point in histochemical detection of tissue copper

The search for a sensitive histochemical method for revealing tissue copper has been the object of many workers in the past. In spite of multiple methods available, the occurrence in clinical practice of negative histochemical stains, even in cases with high copper levels demonstrated by quantitative methods is very high.This study was aimed at verifying the role of technical variations in the sensitivity of the Timm method and, in particular, the role of the dewaxing time of paraffin sections. To this end, 15 liver specimens, 10 from patients affected by Wilson's disease and 5 newborn livers were fixed in 10% formalin, paraffin embedded and routinaly processed. Four 4-micron sections from each case were rinsed in xylene for 10, 20, 60 min, and for 24 hrs. All sections were stained with Timm's method. In 13 out of the 15 liver biopsies utilized in this study, the sensitivity of Timm's method in revealing copper deposits in liver cells appeared to be dependent on the dewaxing time. In two other cases, reactivity of copper granules to Timm solution did not change significantly with the different deparaffination times. The best results were obtained by rinsing sections in xylene for 24 hrs, the worst in sections treated with xylen for 10 minutes. In particular, in five cases of Wilson's disease, Timm stain applied to sections following ten minutes of xylene were completely negative, while copper granules were clearly evidenced in the same section following an overnight bath in xylene. Our data show that an overnight bath of paraffin sections in xylene may completely change the sensitivity of Timm stain in revealing copper deposits in the liver, relaunching copper histochemistry in the diagnosis of copper-related liver diseases.     

Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO₄-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H₂O₂ acidified with 0.1 or 0.01 N H₂SO₄ to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2-0.3% NH₄OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.     

Microwave applications in classical staining methods in formalin-fixed human brain tissue: a comparison between heating with microwave and conventional ovens.

The quality of microwave adaptations of three classical neuroanatomical staining methods (the Nissl, Klüver-Barrera and H?ggqvist stains) was tested on frozen serial sections from human brain specimens which has been stored for up to 10 years in 10% formalin. The conclusion was that the use of microwave irradiation reduces processing time and/or concentrations of the chemicals used, whereas the light microscopical quality of the stains considered is equal or improved as compared to their original counterparts. Next, a comparison was made between microwave adapted stains and classical procedures, which, except for the use of a conventional oven as heat source together with pre-heated solutions, were entirely identical. It appeared, that at light microscopical level no difference can be appreciated between the effect of internally (using microwave irradiation) and externally (using a conventional oven) supplied heat on the staining result.     

A Single Simple Procedure for Dewaxing,Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue

Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin-fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene.In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin-fixed paraffin-embedded tissue.Key words: Immunolabelling, antibodies, histology, protein localization, HIER     

Buffered Romanowsky-Giemsa method for formalin fixed,paraffin embedded sections: taming a traditional stain

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96?100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H &; E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H &; E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H &; E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.     

Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain

The components of the hematoxylin and eosin (H & E) stain (i.e. hemalum and eosin Y), their contributions to the typical staining pattern, and the reasons why the H & E stains are the preferred oversight stains for routine diagnostic histopathology are discussed. The essential diagnostic significance of effective nuclear staining by hemalum, providing information on nuclear morphology and texture, is emphasized; as is the ironic advantage for routine diagnostic histopathology of the limited range of colors provided by H & E staining, that allows recognition of significant features under low microscopic magnifications. Standardization of hemalum is considered, along with probable reasons why users show resistance to such a concept. Counterstaining with anionic (acid) dyes is discussed, as is the important phenomenon of contrast. The particular advantages and disadvantages of eosin Y and phloxin B as counterstains to hemalum are outlined. The concept of an “ideal routine histological stain” is considered, and H & E is compared to such an ideal case. Finally, deficiencies of H & E staining are discussed, and a program to develop an improved oversight stain is introduced.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

Chlorazol Black E as a Stain for Tension Wood

Sections containing gelatinous fibers were cut at 15 μ from material both fixed and stored in formalin-acetic-alcohol, 5:5:90 (of 70%). These sections were stained 5 min in a 1% aqueous solution of lignin pink (G. T. Gurr), differentiated quickly in water, soaked 5 min in 95% ethyl alcohol, dehydrated in absolute ethyl alcohol and counter stained 5 min with a 1% solution of chlorazol black E (G. T. Gurr) in methyl cellosolve, followed by dehydration in absolute ethyl alcohol, clearing in xylene and mounting in Canada balsam. The gelatinous layer was sharply defined as a dense black zone whilst the remainder of the cell wall stained light pink. The specificity of the technique was superior to that of safranin and light green, and was not easily obscured by overstaining. The technique is particularly useful for locating small zones of gelatinous fibres, and for photomicrographical work.     

Microwave Fixation of Whole Fetal Specimens

This study compares microwave fixation of whole fetal specimens with conventional techniques performed at room temperature. All fetuses were obtained from the same pregnant rat; half of them were placed in neutral formalin for 15 min at room temperature, then irradiated for 2.5 min in a domestic microwave oven. The remaining fetuses were placed in neutral formalin at room temperature for 48 hr as a control. Both experimental and control groups were exposed to routine tissue processing for light microscopy and embedded in paraffin wax. Sections 5 μm thick were stained with hematoxylin and eosin. Our results showed that the microwave technique reduced the fixation time while providing thin sections that were equal to or better in quality than those in the control group.