Replacing xylene with n-heptane for paraffin embedding JC Stockert, B López-Arias, P Del Castillo, A Romero, A Blázquez-Castro Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Cantoblanco, Madrid 28049, Spain
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Accepted June 7, 2012
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 hematoxylineosin or Masson’s trichrome showed proper embedment, well preserved morphology and excellent staining. Key words: n-heptane, paraffin, solvents, tissue embedding, xylene
It is well known that aromatic and halogenated hydrocarbon solvents have adverse effects on health. In histology and histopathology laboratories, xylene, toluene or chloroform are widely used for clearing in paraffin embedding and these solvents also are used for de-waxing paraffin sections. The most common mounting media, e.g., DePeX, also contain xylene as solvent. The health hazard associated with using these solvents is an important issue for research and clinical laboratories (Brunton 1992, Coleman 2001). Exposure to aromatic and chlorinated solvents causes damage to the respiratory system, skin, liver, kidney and central nervous system (Langman 1994, Armstrong and Green 2004). The Material Safety Data Sheet (MSDS) for laboratory chemicals should be checked at http://www.proscitech.com/ for handling and use of these and other solvents. The Agency for Toxic Substances and Disease Registry (ATSDR/EPA) recently published a list of chemicals with known or suspected toxicity. Among 275
Correspondence: Dr. Alfonso Blázquez-Castro. Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Darwin 2 Cantoblanco, Madrid 28049, Spain. Telephone: ⫹ 34 91 5854426. e-mail:
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DOI:10.3109/10520295.2012.701764
compounds listed for their toxic effects, chloroform and xylene ranked 11th and 48th, respectively, which indicates a high potential for adverse health effects (http://www.atsdr.cdc.gov/cxcx3.html). During the past few years, substitutes have been employed as clearing and de-waxing agents for paraffin processing of tissues. Maxwell (1978) suggested using trichloroethane as a clearing solvent and this has been employed increasingly (Temel et al. 2005). Although it is not flammable, adequate ventilation is required, because the vapors are toxic (Reid and Young 1981). Other substitutes proposed for xylene include vegetable oils (Bruun Rasmussen et al. 1992), butyldecanoate (Lyon et al. 1995), isopropanol (Viktorov and Proshin 2003), isopropanolmineral oil mixtures (Buesa and Peshkov 2009), and propylene glycol methyl ester (Chen et al. 2010). De-waxing protocols using octane (Fredricks and Relman 1999, Xing et al. 2010) and water-detergent mixtures (Falkeholm et al. 2001) also have been described. Although lacking a precise chemical structure, isoparaffin H, (Panreac, Barcelona) now is sold as a xylene substitute. We propose n-heptane (C7), an aliphatic hydrocarbon, as a substitute for xylene. n-Heptane is a constituent of gasoline, itself a mixture of C4–C12 hydrocarbons, that is used as standard for testing performance and knocking conditions of Ottocycle engines (Windholz 1983). In fact, aliphatic 1
hydrocarbons have been employed previously as solvents for histopathology and histochemistry (Lillie and Fullmer 1976). The solvent employed, known as “white gas,” was a mixture mainly of hexane-heptane hydrocarbons. n-Heptane is an inert, nonfluorescent solvent that can be used as a substitute for xylene and other solvents for paraffin processing of tissues.
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Material and methods Tissue samples from Wistar rats including tongue, salivary glands, esophagus, stomach, large and small intestine, liver, kidney, adrenal, lung and skin were fixed either by perfusion with 3.7% formaldehyde in phosphate buffered saline (PBS), or by immersion in 3.7% formaldehyde or Bouin’s solution at room temperature. After fixation for 30 min, samples were cut into small pieces and left in fresh fixative for 24 h. Specimens were washed in tap water for 24 h and dehydrated in 30, 50, 70, 96 and 100% ethanol for 1 h at each concentration. Clearing was accomplished using two changes of n-heptane (Scharlau Chemie, Sentmenat, Spain; 99% purity) for 10 min taking care that samples always remained at the bottom of the container. As controls, tissue samples also were cleared in xylene and embedded as usual. The tissues cleared in n-heptane were placed in a mixture of paraffin/nheptane (1:1, v/v) at 56° C for 1 h, then embedded in paraffin wax at 56° C for 24 h. Sections were obtained using a Microm HM 355 S-2 microtome (Walldorf, Germany), de-waxed with n-heptane for 15 min and hydrated in descending alcohols (100, 96 and 70% ethanol, 2 min at each concentration) to water. Sections were stained with hematoxylineosin (H-E) and Masson’s trichrome using standard methods. Microscopic observations and photography of sections mounted in DePeX were carried out using a CX-31 RBSF microscope (Olympus, Tokyo, Japan) equipped with an Olympus UC-30 digital camera. Photomicrographs were processed using Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA).
Results Paraffin sections obtained using n-heptane for tissue embedding and de-waxing revealed excellent preservation of morphology; sections were entirely similar to those processed with xylene (Fig. 1A). Figure 1, B and C illustrate the microscopic structure of a kidney section using 2
Fig. 1. Bright field photomicrographs of paraffin sections from rat kidney. Samples were fixed by immersion in 3.7% formaldehyde, processed with xylene (A) or n-heptane (B, C) and stained with H-E. A and B) Part of the cortical renal region at low magnification. C) Detail of a renal glomerulus. Bars ⫽ 30 μm.
n-heptane. Although no quantitative evaluations were made, retractions commonly found in embedded samples after xylene were less conspicuous. Staining by H-E or Masson’s trichrome was identical to that of sections processed with xylene. Sections of other tissues processed with n-heptane showed good morphological preservation of tissues and staining quality.
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Fig. 2. Chemical structure of paraffin wax (A), m-xylene (B) and n-heptane (C), and their corresponding space-filling molecular models from HyperChem 8 software (A,’ B,’ and C,’ respectively). Paraffin wax is the aliphatic chain, C16H34, and commercial xylene is a mixture of the three aromatic isomers (o-, m-, and p-); the p-isomer is the predominant form (Windholz 1983).
Discussion Histology and histopathology laboratories are hazardous work places owing to the abundance of chemicals that can pose major safety and health concerns (Brunton 1992, Langman 1994, Coleman 2001, Armstrong and Green 2004). Xylene and toluene, the most common aromatic solvents used for paraffin embedding in histology and histopathology laboratories are highly toxic. Chloroform and trichloroethane sometimes are used for paraffin processing, but these chlorinated compounds also are harmful (Reid and Young 1981, Armstrong and Green 2004, Temel et al. 2005). Although several alternatives for xylene are available, they are not widely used and other substitutes are necessary. n-Heptane does not have an unpleasant smell nor does it irritate the mucous membranes. As illustrated in Fig. 2, the short
aliphatic chain of n-heptane (Fig. 2C) is far more similar to the paraffin aliphatic chain (Fig. 2A) than the aromatic hydrocarbons xylene and toluene (Fig. 2B). n-Heptane also is relatively inexpensive and it is not so dangerous and toxic as aromatic or chlorinated solvents. Considering the excellent results obtained, n-heptane appears to be a convenient substitute for the usual aromatic solvents used in histological techniques.
Acknowledgment This work was supported by a grant from the Ministerio de Ciencia e Innovación (CTQ2010-20870C03-03/BQU), Spain. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. n-Heptane paraffin embedding 3
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