25-Year Storage of Human Choroid Plexus in Methyl Salicylate Preserves Its Antigen Immunoreactivity
Dina A. SUFIEVA, Elena A. FEDOROVA, Vladislav S. YAKOVLEV, Olga V. KIRIK, Daria L. TSYBA, Igor P. GRIGOREV, Dmitrii E. KORZHEVSKII
Department of General and Specific Morphology, Institute of Experimental Medicine, ST. PETERSBURG, RUSSIA
Keywords: Choroid plexus, Methyl salicylate, Pineal gland, Human, Immunohistochemistry
Immunohistochemical investigation of archival histological material is a serious problem, since long-term storage of biological tissues,
most often in formalin, leads to a loss of antigenic properties. However, the biological material can also be stored in the clearing agent methyl
salicylate. The aim of this study was to assess the antigenicity of the human choroid plexus after extra long-term storage in methyl salicylate.
Material and Method: The study was performed on samples of fixed human choroid plexus (occasionally with attached neighboring pineal
gland) stored in either methyl salicylate or paraffin blocks for 25 years. Chromogenic and fluorescence immunohistochemistry of vimentin,
GFAP, type IV collagen, β-catenin, α-smooth muscle actin, von Willebrand factor, CD68, mast cell tryptase, TMEM119, and synaptophysin was
Results: The storage of human choroid plexus in methyl salicylate for 25 years does not impair its histomorphology and preserves the properties
of all the antigens assessed, which makes their immunohistochemical visualization possible using both light and fluorescence microscopy.
Additionally, we found that long-term storage of human choroid plexus in methyl salicylate does not cause an increase in autofluorescence.
Conclusion: Methyl salicylate can be recommended as a medium for long-term storage of biological tissue, as it provides excellent brain tissue
preservation and retains its antigenic properties for up to 25 years.
Pathological or experimental histological specimens
cannot be processed and examined immediately after
they are obtained in many cases, and they are then are
exposed to long-term storage in a fixative, most often
formalin. As a rule, prolonged storage in the formalin does
not impair the histomorphology of brain specimens, but
the quality of some histological staining is worsened 1
Moreover, prolonged fixation in formalin can result in the
irreversible loss of immunoreactivity for many antigens 1-6
. However, tissue specimens can be preserved long-term
not only in a fixative, but also in methyl salicylate. Methyl
salicylate, the main component of wintergreen oil, is a
commercially available, strong-smelling organic ester that
is used in histological and histopathological laboratories
as a perfect substitute for xylene at the clearing stage of
preparation processing 7-9
. As an excellent clearing
agent, it is especially recommended for bone and muscle
tissues that are adversely affected by conventional clearing
with xylene 10
Previously, it has been shown that prolonged storage (up
to three years) of rat brain specimens in methyl salicylate had no detectable effect on the immunoreactivity of common
markers of normal and cancer brain cells, such as
neuronal nuclear protein, neuron-specific enolase, glial
fibrillary acidic protein (GFAP), vimentin, nestin, and
doublecortin 11. These data encouraged us to conduct
the present study, in which we assess and confirm the suitability
of human choroid plexus samples stored in methyl
salicylate for twenty-five years to routine histological staining
and immunohistochemical revealing of antigens. The
results showed excellent histological staining and good immunohistochemical
visualization of various brain antigens
(vimentin, GFAP, type IV collagen, β-catenin, α-smooth
muscle actin, von Willebrand factor, cluster of differentiation
68 (CD68), mast cell tryptase, transmembrane protein
119 (TMEM119), and synaptophysin) by using both light
and fluorescence microscopy. Fluorescence-based imaging
techniques are often hampered by the background autofluorescence,
which can arise from the endogenous sample
components such as age pigment lipofuscin, elastin and collagen
proteins, serotonin, catecholamines, and some others
12,13. Formaldehyde fixation is also known to induce an
intense autofluorescence in samples of paraffin-embedded
animal and human tissues making the fluorescence microscopic observation and immunofluorescence analysis more
difficult 14,15. Considering the above, we also aimed to
investigate whether prolonged storage in methyl salicylate
causes an increase in autofluorescence in the choroid plexus
The study was conducted in accordance with the
Declaration of Helsinki of 1975, and had been approved by
the local Ethics Committee. Fragments of choroid plexus
(some of them containing adjacent pineal gland) from
the lateral and fourth ventricles were removed at routine
autopsy from 14 persons (12 males and 2 females aged 20-
59) in 1995-1996. The specimens were fixed in alcoholic
formalin for 1-2 days and dehydrated in ethanol series.
Then, some of the samples were embedded in paraffin
routinely, and some were immersed into methyl salicylate
(Vekton, St. Petersburg, Russia) in 20 ml glass containers
and hermetically sealed. The containers were stored in the
laboratory at room temperature. In 2021, after washing
in three portions of absolute alcohol, the samples were
processed in the spin tissue processor (STP 120, Thermo
Fisher Scientific, Waltham, MA, USA) and embedded in
paraffin by a routine procedure. Then, samples of choroid
plexus in paraffin blocks prepared in 1995-96 (immediately
after fixation) and 2021 (after storage in methyl salicylate)
were cut to obtain 7 μm-thick sections using rotary (RM
2125RT, Leica Microsystems, Wetzlar, Germany) or
sliding (Leica SM 2000R, Leica Microsystems, Wetzlar,
Germany) microtome, mounted on poly-L-lysine-coated
(Polysine™, Menzel-Gläser, Braunschweig, Germany) glass
slides. For histological and immunohistochemical staining,
the sections were deparaffinized with xylene, rehydrated
in descending gradient of ethanol, and rinsed in distilled
Hematoxylin and eosin staining was performed according
to the commonly established procedure. For immunohistochemistry,
the heat-induced antigen retrieval was
performed in modified citrate buffer, pH 6.1 (S1700, Agilent-
Dako, Santa Clara, CA, USA) for 25 min at 90ºC. Endogenous
peroxidase was quenched by incubation in 3%
aqueous solution of hydrogen peroxide for 10 minutes. After
washing three times in the phosphate-buffered saline,
pH 7.4, the sections were pretreated with the blocking solution
(Protein Block, Spring Bioscience, Pleasanton, CA,
USA) for 10 min at room temperature. Then, the sections
were incubated with the primary antibodies against the following
antigens: GFAP, vimentin, synaptophysin, CD68,
TMEM119, mast cell tryptase, type IV collagen, α-smooth
muscle actin, and von Willebrand factor. Information regarding
the panel of antibodies tested in this study and
their dilution is presented in Table I. These antigens are
specific markers of astrocytes, microglia, macrophages,
synapses, mast cells, endothelial cells, basement membrane
of vessels, and smooth muscle cells applicable for both normal
and cancer tissues.
Table I. Specification of the primary antibodies used in this study.
To visualize the primary antibodies using light microscopy
the following reagents were applied: for rabbit antibodies,
Reveal Polyvalent HRP DAB (Spring Bioscience, Pleasanton,
CA, USA); for mouse antibodies, MACH 2 Mouse
HRP-Polymer (BioCare Medical, Pacheco, CA, USA). The
peroxidase label was detected using diaminobenzidine
chromogen (DAB+; Agilent-Dako, Santa Clara, CA, USA).
After immunohistochemical reactions, some sections were
counterstained with hematoxylin or alcian blue. For all immunohistochemical
reactions, the control reactions recommended
by the antibody manufacturer were performed.
For detection of the primary antibodies by confocal laser
microscopy the following reagents were applied: for rabbit antibodies, Reveal Polyvalent HRP DAB (Spring Bioscience,
Pleasanton, CA, USA) and anti-HRP Fab-fragment of
goat immunoglobulin conjugated with Cy3 fluorochrome
(1:100, Jackson ImmunoResearch, West Grove, PA, USA)
with emission maximum (λem=569 nm) in red light or anti-
rabbit Fab-fragment of donkey immunoglobulin conjugated
with a Rhodamine Red-X (RRX) fluorochrome (1:50,
Jackson ImmunoResearch, West Grove, PA, USA) with
emission maximum (λem=590 nm) in red light; for mouse
antibodies, biotinylated anti-mouse antibody (VectorLabs,
USA) and streptavidin conjugated with a Rhodamine Red-
X (RRX) fluorochrome (1:150, Jackson ImmunoResearch,
West Grove, PA, USA) with emission maximum (λem=590
nm) in red light. For nuclei counterstaining, SYTOX Green
(1:100, Thermo Fisher Scientific, Waltham, MA, USA) was
used. The stained sections were coverslipped using fastdrying
permanent non-fluorescent mounting medium Cytoseal
™ 60 (Richard-Allan Scientific, Kalamazoo, MI, USA).
For examination of sections in transmitted light, the
Leica DM750 microscope and ICC50 camera (Leica
Microsystems, Wetzlar, Germany) were used. The sections
prepared for confocal laser microscopy were examined
under a LSM800 confocal laser microscope (Carl Zeiss,
Oberkochen, Germany) using Zen-2012 (Carl Zeiss,
Oberkochen, Germany) software for image processing.
The primary assessment of the choroid plexus tissue
was carried out on preparations stained routinely with hematoxylin and eosin (Figure 1
). All tissue samples
showed good preservation without visual manifestations
of autolysis or tissue compression. The cell nuclei were
stained in blue-violet color. The cytoplasm of epithelial
cells of the choroid plexus and the smooth muscle cells in
the blood vessel walls showed moderate oxyphilia. Redbrown
erythrocytes were visible in the lumen of the vessels.
Connective tissue fibers were pink in color. No differences
in tinctorial properties were found between the tissue
samples stored in paraffin blocks or methyl salicylate.
Click Here to Zoom
|Figure 1: Representative figures of human choroid plexus after 25 years of storage in methyl salicylate (A), or paraffin blocks (B). Note
the well-preserved tissue, crispness of the epithelial cells and blood vessels containing red cells in both methyl salicylate- and paraffin
block-stored material. Hematoxylin and eosin staining. x40. Scale bar corresponds to 50 μm.
All antigens studied preserve their immunoreactivity in the
choroid plexus after prolonged storage in methyl salicylate.
The used antibodies label their typical targets. The choroid
epithelial cells were strongly vimentin-immunopositive
and surrounded by β-catenin immunoreactivity (Figure
2A;3D). Immunoreaction for CD68, TMEM119, and mast
cell tryptase labels macrophages, microglia, and mast cells,
respectively (Figure 2C,E;3A). Antibody for α-smooth
muscle actin reveals actin inside vascular smooth muscle
cells, whereas type IV collagen and von Willebrand factor
are concentrated around smooth muscle cells (basal
lamina) and in vascular endothelium, respectively (Figure
3B;3C). GFAP- and synaptophysin-immunoreactivity
was not found in the choroid plexus, but can easily be
revealed in the adjacent pineal gland (Figure 3E;4B). It is
important to note that all immunoreactive structures stain
distinctly in the absence of intense background staining.
Immunoreaction for all markers was the same regardless of
whether it was carried out on the material stored in methyl salicylate or in paraffin blocks. Moreover, the impression
was that immunostaining for type IV collagen and vimentin
was generally more intense in the choroid plexus after
storage in methyl salicylate than in paraffin blocks.
Click Here to Zoom
|Figure 2: Representative figures of
immunostaining for vimentin (A, B),
CD68 (C, D), and mast cell tryptase
(E-F) in the human choroid plexus after
25 years of storage in methyl salicylate
(left column), or paraffin blocks (right
column). Counterstaining: hematoxylin
(A-D) or alcian blue (E-F). x40. Scale
bar: 50 μm (A-D), 20 μm (E-F).
Click Here to Zoom
|Figure 3: Representative figures of immunostaining for TMEM119 (A), type IV collagen (B), von Willebrand factor (C), and β-catenin
(D) in the human choroid plexus and synaptophysin (E) in the human pineal gland after 25 years of storage in methyl salicylate. Note
the excellent definition of immunostained structures. Counterstaining: A – none, B, D, E – hematoxylin, C – alcian blue. x40. Scale bar:
Click Here to Zoom
|Figure 4: Immunofluorescent imaging of type IV collagen in the human choroid plexus (A) and GFAP in the human pineal gland
(B) after 25 years of storage in methyl salicylate. A) type IV collagen (red) and nuclear counterstaining with SYTOX Green (green).
B) GFAP (red) and nuclear counterstaining with SYTOX Green (green). Confocal laser microscopy. x240 (A), x40 (B). Scale bar: 5
μm (A), 20 μm (B).
Confocal Laser Microscopy
Fluorescent immunohistochemistry is also applicable to
methyl salicylate-stored material. Representative confocal
microscopic image of type IV collagen immunoreactivity
is shown in Figure 4A. All markers used were visualized
by immunofluorescence as clearly and distinctly as by
light microscopic immunohistochemistry. No significant autofluorescence was observed. Similarly, a high quality
immunofluorescent label was seen in the pineal gland
adjacent to the choroid plexus (Figure 4B).
The obtained results show for the first time that samples
of human choroid plexus and pineal gland preserve their
antigenicity after 25-year storage in methyl salicylate. This
material is suitable for full-fledged immunohistochemical
study of various markers using both light and fluorescence
microscopy. The antibodies used in the present study
clearly label their typical target structures with minimal background.
The localization of vimentin, CD68, tryptase,
α-smooth muscle actin, and type IV collagen in the human
choroid plexus exposed to prolonged storage in methyl
salicylate was similar to that described previously in freshly
prepared human choroid plexus 16-20
To our knowledge, no prior studies have documented
the presence of TMEM119 and von Willebrand factor in
the choroid plexus of humans or experimental animals
(despite an intensive search for TMEM119 in the murine
choroid plexus 21). Thereby, this is the first report on
the localization of TMEM119 and von Willebrand factor
in choroid plexus. β-Catenin has been described in the
choroid plexus of rats, but not in humans 22. Our present
observation has shown that its distribution in humans is
about the same as in rats.
We failed to detect GFAP- and synaptophysin-immunopositive
structures in the human choroid plexus. The absence of GFAP expression in the choroid plexus is
consistent with the results of other researchers 18. As for
synaptophysin, to the best of our knowledge, there are no
data on its expression in the choroid plexus. To confirm
that the lack of GFAP and synaptophysin is not due to
poor antibody quality or improper immunohistochemical
technique used, we examined the immunoreactivity of
GFAP and synaptophysin in sections of pineal gland
tissue presented in some choroid plexus specimens.
Well-discernible numerous GFAP- and synaptophysinimmunopositive
profiles can be identified in the human
pineal gland using the same technique. Therefore, the
obtained results indicate the absence of GFAP and
synaptophysin expression in the human choroid plexus.
Comparison of material stored in methyl salicylate or in
paraffin blocks for twenty-five years revealed an insignificant
difference in the intensity and specificity of their
immunostaining. Moreover, in some cases, the methyl salicylate-stored choroid plexus appears to show better visualization
of the immunohistochemical markers used than
The results obtained demonstrate that not only chromogenic
immunohistochemistry, but also fluorescent immunohistochemistry
is applicable to choroid plexus samples
after long-term storage in methyl salicylate. This material
exhibits no signs of increase in background autofluorescence,
which is the major drawback to the acquisition of
clear images in fluorescent immunohistochemistry. Our
observations show that storage of choroid plexus samples
in methyl salicylate does not impair the quality of images
in a fluorescence microscope in any way.
Currently, formalin is widely used in morphological and
pathological laboratories as a conventional fixative and a
universal preservative for a long-term storage of histologic
material. However, formaldehyde is classified by the
International Agency for Research on Cancer as a definitive
human carcinogen (Group A1) and poses a significant threat
to human health 23,24. This is a significant disadvantage
of formalin usage in the laboratory. In comparison to
formalin, methyl salicylate is significantly less dangerous
for humans: it is harmful if swallowed and may irritate
eyes or skin, but this is an inherent property of most other
chemicals routinely used in histological and pathological
laboratories 25. Moreover, methyl salicylate is used in the
clinic as a topical analgesic and anti-inflammatory agent 26. Therefore, the use of methyl salicylate is much safer
than the use of formalin.
In addition, prolonged storage of biological samples in
formalin can lead to irretrievable loss of many antigens
making such material unsuitable for immunohistochemical
investigation 1-6. On the contrary, the use of methyl
salicylate, according to the data presented here, retains the
antigenicity in at least the choroid plexus and the pineal
gland after long-term storage. This claim is supported by
finding for the first time of TMEM119-, von Willebrand
factor- and β-catenin-immunoreactive structures in the
human choroid plexus stored in methyl salicylate for 25
Previously, good preservation of the immunoreactivity of
a number of antigens has been demonstrated in rat brain
samples after prolonged (up to 3 years) storage in methyl
salicylate 11. Thereby, methyl salicylate is preferred
over formalin if long-term storage of either human or rat
brain specimens is required, as it is safe and preserves the
immunoreactivity of antigens in brain tissue.
The present study demonstrates for the first time that storage
of human choroid plexus and pineal gland in methyl
salicylate for 25 years has no detectable influence on histomorphology
and quality of standard histological staining.
The results obtained show good immunohistochemical visualization
of various brain antigens (vimentin, GFAP, type IV collagen, β-catenin, α-smooth muscle actin, von Willebrand
factor, CD68, mast cell tryptase, synaptophysin, and
TMEM119) by using both light and fluorescence microscopy.
Storage in methyl salicylate for 25 years does not intensify
the background autofluorescence in human choroid
plexus and pineal samples and, thereby, does not impair
the quality of immunofluorescence. Therefore, methyl salicylate
can be recommended to preserve antigenicity and
suitability for histological and immunohistochemical study
of the stored material when long-term storage of brain tissue
samples is needed.
Conflict of Interest
The research was conducted in the absence of any financial, personal,
academically competitive or intellectual relationships that could be
construed as a potential conflict of interest.
The study was conducted within the state assignment of the Institute
of Experimental Medicine.
Concept: DEK, Design: DEK, Data collection or processing: DAS,
EAF, VSY, OVK, DLT, DEK, Analysis or Interpretation: OVK, IPG,
DEK, Literature search: IPG, DEK, Writing: IPG, DEK, Approval:
DAS, EAF, VSY, OVK, DLT, IPG, DEK.
1) Sanderson T, Wild G, Cull AM, Marston J, Zardin G.
Immunohistochemical and immunofluorescent techniques. In:
Suvarna KS, Layton C, Bancroft JD, editors. Bancroft’s theory
and practice of histological techniques. 8th ed. Philadelphia:
Churchill Livingstone Elsevier; 2019. 337-94.
2) Hayat MA. Microscopy, immunohistochemistry, and antigen
retrieval methods for light and electron microscopy. New York:
Kluwer Academic Publishers. 2002.
3) Hoetelmans RW, Prins FA, Cornelese-ten Velde I, van der Meer
J, van de Velde CJ, van Dierendonck JH. Effects of acetone,
methanol, or paraformaldehyde on cellular structure, visualized
by reflection contrast microscopy and transmission and scanning
electron microscopy. Appl Immunohistochem Mol Morphol.
4) Merrell GA, Troiano NW, Coady CE, Kacena MA. Effects of
long-term fixation on histological quality of undecalcified murine
bones embedded in methylmethacrylate. Biotech Histochem.
5) Webster JD, Miller MA, Dusold D, Ramos-Vara J. Effects of
prolonged formalin fixation on diagnostic immunohistochemistry
in domestic animals. J Histochem Cytochem. 2009;57:753-61.
6) Webster JD, Miller MA, DuSold D, Ramos-Vara J. Effects of
prolonged formalin fixation on the immunohistochemical
detection of infectious agents in formalin-fixed, paraffinembedded
tissues. Vet Pathol. 2010;47:529-35.
7) Jongedijk E. A rapid methyl salicylate clearing technique for
routine phase contrast observations on female meiosis in
Solanum. J Microscopy. 1987;146:157-62.
8) Karnam S, Girish HC, Murgod S, Nayak VN, Varsha VK,
Yanduri S. Rapid tissue processing technique: A novel method
using methyl salicylate. J Oral Maxillofac Pathol. 2018;22:443.
9) Niederegger S, Wartenberg N, Spiess R, Mall G. Simple clearing
technique as species determination tool in blowfly larvae.
Forensic Sci Int. 2011;206:e96-8.
10) Skinner RA. The value of methyl salicylate as a clearing agent. J
11) Korzhevskii DE, Gilyarov AV. Immunocytochemical detection
of tissue antigens after prolonged storage of specimens in
methylsalicylate. Neurosci Behav Physiol. 2010;40:107-9.
12) Banerjee B, Miedema BE, Chandrasekhar HR. Role of basement
membrane collagen and elastin in the autofluorescence spectra of
the colon. J Investig Med. 1999;47:326-32.
13) Del Castillo P, Llorente AR, Stockert JC. Influence of fixation,
exciting light and section thickness on the primary fluorescence
of samples for microfluorometric analysis. Basic Appl Histochem.
14) Baschong W, Suetterlin R, Laeng RH. Control of autofluorescence
of archival formaldehyde-fixed, paraffin-embedded tissue in
confocal laser scanning microscopy (CLSM). J Histochem
15) Davis AS, Richter A, Becker S, Moyer JE, Sandouk A,
Skinner J, Taubenberger JK. Characterizing and diminishing
autofluorescence in formalin-fixed paraffin-embedded human
respiratory tissue. J Histochem Cytochem. 2014;62:405-23.
16) Fernández-Sevilla LM, Valencia J, Flores-Villalobos MA,
Gonzalez-Murillo Á, Sacedón R, Jiménez E, Ramírez M, Varas A,
Vicente Á. The choroid plexus stroma constitutes a sanctuary for
paediatric B-cell precursor acute lymphoblastic leukaemia in the
central nervous system. J Pathol. 2020;252:189-200.
17) Fedorova EA, Grigorev IP, Syrtzova MA, Sufieva DA, Novikova
AD, Korzhevskii DE. Detection of morphological signs of mast
cell degranulation in the human choroid plexus using different
staining methods. Morfologiia. 2018;153:70-5. (In Russian).
18) Sarnat HB. Histochemistry and immunocytochemistry of the
developing ependyma and choroid plexus. Microsc Res Tech.
19) Vercellino M, Votta B, Condello C, Piacentino C, Romagnolo
A, Merola A, Capello E, Mancardi GL, Mutani R, Giordana
MT, Cavalla P. Involvement of the choroid plexus in multiple
sclerosis autoimmune inflammation: A neuropathological study.
J Neuroimmunol. 2008;199:133-41.
20) Wakamatsu K, Chiba Y, Murakami R, Matsumoto K,
Miyai Y, Kawauchi M, Yanase K, Uemura N, Ueno M.
Immunohistochemical expression of osteopontin and
collagens in choroid plexus of human brains. Neuropathology.
21) Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian
JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A,
Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG,
Barres BA. New tools for studying microglia in the mouse and
human CNS. Proc Natl Acad Sci U S A. 2016;113:E1738-46.
22) Kirik OV, Sufieva DA, Nazarenkova AV, Korzhevskii DE. Cell
contact protein β-catenin in ependymal and epithelial cells in
the choroid plexus of the lateral ventricles of the brain. Neurosci
Behav Physiol. 2017;47:117-21.
23) Cogliano VJ, Grosse Y, Baan RA, Straif K, Secretan MB,
El Ghissassi F; Working Group for Volume 88. Meeting
report: Summary of IARC monographs on formaldehyde,
2-butoxyethanol, and 1-tert-butoxy-2-propanol. Environ Health
24) Santovito A, Schilirò T, Castellano S, Cervella P, Bigatti
MP, Gilli G, Bono R, DelPero M. Combined analysis of
chromosomal aberrations and glutathione S-transferase M1 and
T1 polymorphisms in pathologists occupationally exposed to
formaldehyde. Arch Toxicol. 2011;85:1295-302.
25) Safety data for methyl salicylate Archived 2007-10-13 at
the Wayback Machine, Physical & Theoretical Chemistry
Laboratory, Oxford University. https://web.archive.org/
methyl_salicylate.html [Last accessed on 2022 March 20].
26) Jurca T, Józsa L, Suciu R, Pallag A, Marian E, Bácskay I, Mureșan
M, Stan RL, Cevei M, Cioară F, Vicaș L, Fehér P. Formulation
of topical dosage forms containing synthetic and natural antiinflammatory
agents for the treatment of rheumatoid arthritis.
Copyright © 2022 The Author(s). This is an open-access article published by the Federation of Turkish Pathology Societies under the terms of the Creative Commons Attribution License
that permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No use, distribution, or reproduction is permitted that does not comply with these terms.