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.     

Comparison of verdeluz orange G and modified Gallego stains

Tumors of the oral cavity include combinations of hard and soft tissues that may be difficult to identify using routine hematoxylin and eosin (H & E) staining. Although combination stains can demonstrate hard and soft tissues, trichrome stains, such as VanGieson and Masson, cannot differentiate dental hard tissues, such as dentin, cementum and osteoid. Modified Gallegos (MGS) and verdeluz orange G-acid fuchsin (VOF) stains can differentiate components of teeth. We used 10 tissue sections of decalcified bone and 10 pathologic tissue sections that contained different calcified tissues including peripheral ossifying fibroma, odontoma, central ossifying fibroma and cemento-ossifying fibroma. Sections were stained with H & E, VOF or MGS. H and E stained both hard tissues pink. VOF stained bone purple-red, cementum red and collagen blue. MGS stained bone green-blue, cementum red and collagen blue. VOF staining intensity and differentiation was better than MGS staining. VOF staining demonstrated hard tissue components distinctly and exhibited good contrast with the surrounding connective tissue. VOF also is a simple, single step, rapid staining procedure.     

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.     

A novel xylene substitute for histotechnology and histochemistry

Abstract

Propylene glycol methyl ether (PGME) exhibits excellent solvent and coupling properties. A toxicity database provided evidence suggesting that PGME might be a useful substitute for xylene in histotechnology and histochemistry applications. Tissue specimens were fixed, cleared in either PGME or xylene, embedded in paraffin wax, then dewaxed in either PGME or xylene. Sections were treated with the following stains: hematoxylin & eosin (H & E), three special stains of the Gordon/Sweet silver staining method, PAS, and Masson's trichrome, and immunostains including actin, CD3, CD34, CK, CK7/CK9, Ki-67, and ER/PR. The sections were mounted in a resinous medium consisting of PGME and pinene copolymer, then examined under a microscope. Variables such as water tolerance, dimension change, organic solvency, and anti-fading efficacy also were assessed. Depending on the application, PGME performance was equal to or exceeded that of xylene. PGME provided better optical clarity and nuclear detail, did not harden the tissue samples, conserved tissue antigenicity, and was amenable to resinous mounting. Tissues not dehydrated with absolute ethanol also were processed properly. Tissues treated with PGME did not warp or contract compared to those treated with xylene (p < 0.0001). PGME, however, exhibited less organic solvency than xylene. There was no discernible change in the colors of stains in sections processed with PGME even after storage for two years. These results suggest that PGME is a novel xylene substitute for applications in histotechnology and histochemistry.     

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.     

A histochemical comparison of methyl green-pyronin,and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma,oral leukoplakia,oral submucous fibrosis and normal oral mucosa

Abstract

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.     

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 simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

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.     

Effects of pertussis and cholera toxin on the interferon-γ stimulated immunocytochemical staining of ICAM-1 and inositol phosphate formation in a human renal carcinoma cell line

This report describes a new method using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to visualize live viable tissue at the microscopic level. The visualization of the MTT stained tissue provides a metabolic assessment of tissue damage, and can be utilized as an extension of conventional hematoxylin-eosin (H & E) staining. In this report, several tissues were studied with MTT and H & E staining after incisions had been made by a variety of surgical dissecting instruments. A marked improvement in the detection of tissue damage was seen using MTT, regardless of how the damage was caused, i.e., physical, heat, or photon energy. In addition, a distinct zone of damage not noted on conventionally prepared and stained tissues is readily apparent. Thus MTT staining will have utility in both clinical and research studies, concerned with assessing the viability of tissues.     

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     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.