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 carried out.

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.

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 samples.

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.

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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.

Immunohistochemistry
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.

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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).

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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: 20 μm.

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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).

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 paraffin-stored material.

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 years.

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.

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.

Funding
The study was conducted within the state assignment of the Institute of Experimental Medicine.

Authorship Contributions
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. 2001;9:346-51.

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. 2005;80:139-46.

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 Histotech. 1986;9:27-8.

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. 1989;33:251-7.

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 Cytochem. 2001;49:1565-72.

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. 1998;41:14-28.

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. 2022;42:117-25.

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 Perspect. 2005;113:1205-8.

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/ web/20071013225653/http://physchem.ox.ac.uk/MSDS/ME/ 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. Molecules. 2020;26:24.