See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/230594164

Replacing xylene with n -heptane for paraffin embedding

Article  in  Biotechnic & Histochemistry · August 2012

DOI: 10.3109/10520295.2012.701764 · Source: PubMed

CITATIONS READS

9 2,318

5 authors, including:

Begoña López-Arias Pedro ALVAREZ Del Castillo

Universidad Autónoma de Madrid Universidad Autónoma de Madrid
24 PUBLICATIONS   295 CITATIONS    40 PUBLICATIONS   362 CITATIONS   

SEE PROFILE SEE PROFILE

Alfonso Blázquez-Castro
Universidad Autónoma de Madrid
95 PUBLICATIONS   1,422 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Amyloid peptide effect on Drosophila neuromuscular junction View project

Fluorescence Microscopy in Life Sciences View project

All content following this page was uploaded by Alfonso Blázquez-Castro on 05 June 2014.

The user has requested enhancement of the downloaded file.

 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

Accepted June 7, 2012

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clear-
Biotech Histochem Downloaded from informahealthcare.com by University of Glasgow on 10/02/12

ing 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.

Key words: n-heptane, paraffin, solvents, tissue embedding, xylene

For personal use only.

It is well known that aromatic and halogenated
hydrocarbon solvents have adverse effects on
health. In histology and histopathology laborato-
ries, xylene, toluene or chloroform are widely used
for clearing in paraffin embedding and these sol-
vents 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 chemi-
cals 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: alfonso.blazquez@uam.es
© 2012 The Biological Stain Commission
Biotechnic & Histochemistry 2012, 87(7): 464–467.
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) sug-
gested 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), iso-
propanol (Viktorov and Proshin 2003), isopropanol-
mineral 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 hydro-
carbon, 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 Otto-
cycle engines (Windholz 1983). In fact, aliphatic

DOI:10.3109/10520295.2012.701764 464
 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 paraf-
fin processing of tissues.

Material and methods

Tissue samples from Wistar rats including tongue,

salivary glands, esophagus, stomach, large and
small intestine, liver, kidney, adrenal, lung and
Biotech Histochem Downloaded from informahealthcare.com by University of Glasgow on 10/02/12

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;
For personal use only.

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/n-
heptane (1:1, v/v) at 56° C for 1 h, then embed-
ded 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 hematoxylin-
eosin (H-E) and Masson’s trichrome using standard
methods. Microscopic observations and photogra- Fig. 1. Bright field photomicrographs of paraffin sections
phy of sections mounted in DePeX were carried out from rat kidney. Samples were fixed by immersion in 3.7%
using a CX-31 RBSF microscope (Olympus, Tokyo, formaldehyde, processed with xylene (A) or n-heptane (B,
Japan) equipped with an Olympus UC-30 digital C) and stained with H-E. A and B) Part of the cortical renal
camera. Photomicrographs were processed using region at low magnification. C) Detail of a renal glomerulus.
Adobe Photoshop 8.0 software (Adobe Systems, Bars ⫽ 30 μm.
San Jose, CA).

Results n-heptane. Although no quantitative evalua-

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
tions were made, retractions commonly found in
embedded samples after xylene were less conspic-
uous. 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 tis-
sues and staining quality.

n-Heptane paraffin embedding 465

Biotech Histochem Downloaded from informahealthcare.com by University of Glasgow on 10/02/12
For personal use only.

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 aliphatic chain of n-heptane (Fig. 2C) is far more

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 tolu-
ene, the most common aromatic solvents used for
paraffin embedding in histology and histopathol-
ogy 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 sub-
stitutes are necessary. n-Heptane does not have an
unpleasant smell nor does it irritate the mucous
membranes. As illustrated in Fig. 2, the short
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 chlo-
rinated 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 Min-
isterio de Ciencia e Innovación (CTQ2010-20870-
C03-03/BQU), Spain.
Declaration of interest: The authors report no con-
flicts of interest. The authors alone are responsible
for the content and writing of the paper.

466 Biotechnic & Histochemistry 2012, 87(7): 464–467

 References
Armstrong SR, Green LC (2004) Chlorinated
hydrocarbon solvents. Clin. Occup. Environ. Med. 4:
481–496.
Brunton WA (1992) Safety in the laboratory. J. Clin. Pathol.
45: 949–951.
Bruun Rasmussen B, Norring Hjort K, Mellerup I,
Sether G, Christensen N (1992) Vegetable oils instead of
xylene in tissue processing. APMIS 100: 827–831.
Buesa RJ, Peshkov MV (2009) Histology without xylene.
Ann. Diagn. Pathol. 13: 246–256.
Chen CY, He T, Mao XL, Friis TE, Qin RH, Jian YT (2010)
A novel xylene substitute for histotechnology and histo-
chemistry. Biotech. & Histochem. 85: 231–240.
Coleman R (2001) Safety in the histochemistry laboratory.
Biotech Histochem Downloaded from informahealthcare.com by University of Glasgow on 10/02/12
Acta Histochem. 103: 253–260.
Falkeholm L, Grant CA, Magnusson A, Möller E (2001)
Xylene-free method for histological preparation: a multi-
centre evaluation. Lab. Invest. 81: 1213–1221.
Fredricks DN, Relman DA (1999) Paraffin removal from
tissue sections for digestion and PCR analysis. Biotech-
niques 26: 198–200.
Langman JM (1994) Xylene: its toxicity, measurement of
exposure levels, absorption, metabolism and clearance.
Pathology 26: 301–309.
For personal use only.
View publication stats
Lillie RD, Fullmer HM (1976) Histopathologic Technic and
Practical Histochemistry. 4th ed., McGraw-Hill Book Co.,
New York.
Lyon H, Holm I, Prentø P, Balslev E (1995) Non-hazardous
organic solvents in the paraffin-embedding technique: a
rational approach. Aliphatic monoesters for clearing and de-
waxing: butyldecanoate. Histochem. Cell Biol. 103: 263–269.
Maxwell MH (1978) Safer substitutes for xylene and
propylene oxide in histology, haematology, and electron
microscopy. Med. Lab. Sci. 35: 401–403.
Reid KJ, Young FJ (1981) Are trichloroethane-based sub-
stitutes safer than xylene? Med. Lab. Sci. 38: 145–149.
Temel SG, Noyan S, Cavusoglu I, Kahveci Z (2005) A sim-
ple and rapid microwave-assisted hematoxylin and eosin
staining method using 1,1,1 trichloroethane as a de-waxing
and a clearing agent. Biotech. & Histochem. 80: 123–132.
Viktorov IV, Proshin SS (2003) Use of isopropyl alcohol
in histological assays: dehydration of tissue, embedding
into paraffin, and processing of paraffin sections. Bull.
Exp. Biol. Med. 136: 105–106.
Windholz M (1983) The Merck Index. An Encyclopedia of
Chemicals, Drugs, and Biologicals. 10th ed., Merck & Co.,
Rahway, NJ. pp. 4551, 9890–9891.
Xing M, Du Y, Wang X, Niu L, Chen X (2010) A simplified
paraffin embedding method for small botanical samples.
Biotech. & Histochem. 85: 241–246.
n-Heptane paraffin embedding 467