Application of Several Special Staining Methods for Paraffin Sections on Epon-Embedded Semithin Sections

Yıl 2023, Cilt: 25 Sayı: 3, 251 - 256, 30.12.2023

Ayşe Akbaş Senem Esin Yavaş Semiha Ersoy Doruk Başar

https://doi.org/10.18678/dtfd.1318076

Öz

Aim: This study aimed to compare several specific staining protocols recommended for paraffin sections and toluidine blue and light green double staining combination to be tried for the first time with routine toluidine blue staining on semithin epon sections.
Material and Methods: Samples of 1x1x1 mm were taken from the liver, skin, and aorta tissues of Wistar albino adult rats. Tissue samples were fixed with 5% glutaraldehyde at +4º C overnight, postfixed with 1% osmium tetroxide for one hour, and then, blocked with Epon 812 after processing. Semithin sections of 1 μm thickness were obtained from the epon blocks. Sections were stained with Altmann’s method (for mitochondria), Verhoeff’s method (for elastic fibers), Gordon&Sweets’ silver impregnation method (for type III collagen), toluidine blue and light green double staining combination (for type I collagen) and routine toluidine blue method.
Results: In liver sections, mitochondria in hepatocytes were differentiated by the Altmann method, and stromal type III collagen fibers were distinguished with Gordon&Sweets’ method. Elastic lamellar structures were easily observed in black in the aortic sections stained with the Verhoeff method. Successful results were obtained in the staining of dermal type I collagen with toluidine blue and light green double staining in skin sections.
Conclusion: Since the specific staining tried for the first time gave positive results in epon sections, it was concluded that these methods can be used to determine the localization of cellular and intercellular components that are aimed to be examined at the ultrastructural level.

Anahtar Kelimeler

epon sections, special stainings, light green

Yarı-İnce Epon Kesitlere Parafin Kesitler İçin Önerilen Çeşitli Özel Boyama Yöntemlerinin Uygulanması

Öz

Amaç: Bu çalışmada, parafin kesitler için önerilen çeşitli spesifik boyama protokollerinin ve ilk kez denenecek toluidin mavisi ve açık yeşil ikili boyama kombinasyonunun yarı-ince epon kesitlerde rutin toluidin mavi boyamasıyla karşılaştırılması amaçlandı.
Gereç ve Yöntemler: Wistar albino türü erişkin sıçanlara ait karaciğer, deri ve aort dokularından 1x1x1 mm boyutlarında örnekler alındı. Alınan doku örnekleri %5 gluteraldehit ile bir gece süreyle +4º C’de fikse edildi, ardından bir saat %1 osmiyum tetroksit ile postfiksasyon uygulandı ve takip işlemi sonrası Epon 812 ile bloklandı. Epon bloklardan 1 μm kalınlıkta yarı-ince kesitler elde edildi. Kesitler Altmann yöntemi (mitokondri için), Verhoeff yöntemi (elastik lifler için), Gordon&Sweet gümüşleme yöntemi (tip III kollajen için), toluidin mavisi ve açık yeşil ikili boyama kombinasyonu (tip I kollajen için) ve rutin toluidin mavisi yöntemi ile boyandı.
Bulgular: Karaciğer kesitlerinde, Altmann yöntemi ile hepatositlerdeki mitokondriler, Gordon&Sweets yöntemi ile stromal tip III kollajen lifler belirgin bir şekilde ayırt edildi. Verhoeff yöntemi ile boyanan aort kesitlerinde elastik lamellar yapılar siyah renkte kolaylıkla izlendi. Deri kesitlerinde toluidin mavisi ve açık yeşil ikili boyaması ile dermal tip I kollajenin boyanmasında başarılı sonuçlar elde edildi.
Sonuç: İlk kez denenen spesifik boyanmalar epon kesitlerde olumlu sonuçlar verdiği için, bu metodlardan ultrastrüktürel düzeyde incelenmesi hedeflenen hücresel ve hücrelerarası bileşenlerin lokalizasyonunun belirlenmesinde yararlanılabileceği sonucuna varılmıştır.

Anahtar Kelimeler

epon kesit, özel boyamalar, light green

Kaynakça

• Hayat MA. Chemical fixation. In: Hayat MA, editor. Principles and techniques of electron microscopy. 4th ed. Cambridge, UK: Cambridge University Press; 2000. p.4-61.
• Horobin RW. Staining plastic sections: a review of problems, explanations and possible solutions. J Microsc. 1983;131(Pt 2):173-86.
• Woods AE, Stirling JW. Transmission electron microscopy. In: Suvarna SK, Layton C, Bancroft JD, editors. Bancroft’s theory and practice of histological techniques. 7th ed. China: Churchill Livingstone Elsevier; 2013. p.493-538.
• Hand NM. Plastic embedding for light microscopy. In: Suvarna SK, Layton C, Bancroft JD, editors. Bancroft’s theory and practice of histological techniques. 7th ed. China: Churchill Livingstone Elsevier; 2013. p.139-55.
• Hayat MA. Rinsing, dehydration, and embedding. In: Hayat MA, editor. Principles and techniques of electron microscopy. 4th ed. Cambridge, UK: Cambridge University Press; 2000. p.85-137.
• Hayat MA. Staining of semithin sections. In: Hayat MA, editor. Principles and techniques of electron microscopy. 4th ed. Cambridge, UK: Cambridge University Press; 2000. p.360-6.
• Clark G. Staining procedures. 4th ed. London: Williams & Wilkins, Baltimore; 1981.
• Green FJ. The Sigma-Aldrich handbook of stains, dyes, and indicators. 2nd ed. Milwaukee, Wisconsin: Aldrich Chemical Company; 1991.
• Akbaş A, Yavaş SE, Ersoy S, Usta Ç. The effect of different epon solvents on staining of semithin sections. Uludağ Tıp Derg. 2023;49(1):101-7. Turkish.
• Drury RAB, Wallington EA. Carleton’s histological technique. 4th ed. New York: Oxford University Press; 1967.
• Bancroft JD, Layton C. Connective and mesenchymal tissues with their stains. In: Suvarna SK, Layton C, Bancroft JD, editors. Bancroft’s theory and practice of histological techniques. 7th ed. China: Churchill Livingstone Elsevier; 2013. p. 187-214.
• Trump BF, Smuckler EA, Benditt EP. A method for staining epoxy sections for light microscopy. J Ultrastruct Res. 1961;5:343-8.
• Raimondo S, Fornaro M, Di Scipio F, Ronchi G, Giacobini-Robecchi MG, Geuna S. Chapter 5: Methods and protocols in peripheral nerve regeneration experimental research: part II-morphological techniques. Int Rev Neurobiol. 2009;87:81-103.
• Carriel V, Garzon I, Alaminos M, Campos A. Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach. Histochem Cell Biol. 2011;136(6):709-17.
• Carriel V, Garzon I, Alaminos M, Cornelissen M. Histological assessment in peripheral nerve tissue engineering. Neural Regen Res. 2014;9(18):1657-60.
• Alturkistani HA, Tashkandi FM, Mohammedsaleh ZM. Histological stains: A literature review and case study. Glob J Health Sci. 2015;8(3):72-9.
• Motoda M, Matsubara T, Tanei ZI, Sakashita Y, Yamazaki M, Kawakami I, et al. Morphological study of the phrenic nerve to determine a reference value for the myelinated fiber density in elderly individuals. Neuropathology. 2023;43(2):129-34.
• Ghnenis AB, Czaikowski RE, Zhang ZJ, Bushman JS. Toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology. J Vis Exp. 2018;137:58031.
• Wang Q, Duan X, Dong M, Sun S, Zhang P, Liu F, et. al. Clinical feature and sural biopsy study in nitrous oxide-induced peripheral neuropathy. PloS One. 2022;17(9):e0274765.
• Soontrapa P, Dyck PJ, Dyck PJB, Klein CM, Engelstad JN, Davies J, et al. Sural nerve biopsy utility by masked assessment of individual histologic preparations. J Peripher Nerv Syst. 2023;28(2):191-201.
• Rau CS, Kuo PJ, Wu SC, Huang LH, Lu TH, Wu YC, et al. Enhanced nerve regeneration by exosomes secreted by adipose-derived stem cells with or without FK506 stimulation. Int J Mol Sci. 2021;22(16):8545.
• Zhang Z, Wang J, Song Z, Wang Y, Cheng Z, Guo Q, et al. Downregulation of microRNA-199a-5p alleviated lidocaine-induced sensory dysfunction and spinal cord myelin lesions in a rat model. Toxicol Lett. 2021;336:1-10.
• Tanida I, Yamaguchi J, Suzuki C, Kakuta S, Uchiyama Y. Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells. Heliyon. 2023;9(6):e17394.
• Hryn V, Kostylenko Y, Svintsytska N, Bilash V, Lytovka V. The issue of histological identification of M-cells in the Peyer's patches of albino rat small intestine. Wiad Lek. 2022;75(5 pt 2):1309-12.
• Etcharren V, Mouguelar H, Aguilar Valenciano JJ. Identification of telocytes in the oviduct of the mare. Theriogenology. 2023;205:18-26.
• Gayoso J, Garrosa M, Gayoso S, Rodríguez-Arias CA, Martin-Ferrero MA, Gayoso MJ. Three-sectioning method: A procedure for studying hard tissues and large pieces under light and electron microscopy. Micron. 2020;132:102841.
• Petrovic B, Pantelinac J, Capo I, Miljkovic D, Popovic M, Penezic K, et al. Using histological staining techniques to improve visualization and interpretability of tooth cementum annulation analysis. Int J Morphol. 2021;39(1):216-21.
• Krenacs T, Bagdi E, Stelkovics E, Bereczki L, Krenacs L. How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies. J Clin Pathol. 2005;58(9):897-903.
• Aoki A, Gutierrez LS. A simple toluidine blue-basic fuchsin stain for spermatozoa in epoxy sections. Stain Technol. 1967;42(6):307-10.
• Aparicio SR, Marsden P. A rapid methylene blue-basic fuchsin stain for semi-thin sections of peripheral nerve and other tissues. J Microsc. 1969;89(1):139-41.
• Van Reempts J, Borgers M. A simple polychrome stain for conventionally fixed epon-embedded tissues. Stain Technol. 1975;50(1):19-23.
• Oldmixon EH. Mallory's phloxine B-methylene blue-azure II stain emphasizes elastin and collagen bundles in epoxy embedded lung. Stain Technol. 1988;63(3):165-70.
• Fritsch H. Staining of different tissues in thick epoxy resin-impregnated sections of human fetuses. Stain Technol. 1989;64(2):75-9.
• Tato A, Planes M, Ramos A, Stockert JC, Ferrer JM. Differential staining of mucin granules from epoxy resin sections by a phosphotungstic acid-methyl green procedure. Biotech Histochem. 1991;66(3):139-44.
• Cannon MS, McGuinn D, Stuth NR. Combined microwave stain for plastic embedded sections of nerve and connective tissue. J Histotechnol. 1992;15(2):113-5.
• Scala C, Preda P, Cenacchi G, Martinelli GN, Manara GC, Pasquinelli G. A new polychrome stain and simultaneous methods of histological, histochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues. Histochem J. 1993;25(9):670-7.
• Tolivia J, Navarro A, Tolivia D. Polychromatic staining of epoxy semithin sections: a new and simple method. Histochemistry. 1994;101(1):51-5.
• Menovsky T, van den Bergh Weerman M. A modified staining method for connective tissue in semi-thin histological sections of peripheral and cranial nerves. Acta Chir Plast. 1999;41(4):120-2.
• D'Amico F. A polychromatic staining method for epoxy embedded tissue: a new combination of methylene blue and basic fuchsine for light microscopy. Biotech Histochem. 2005;80(5-6):207-10.
• Morikawa S, Sato A, Ezaki T. A simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections. Microscopy (Oxf). 2018;67(6):331-44.
• Manskikh VN, Sheval EV. An adaptation of Twort’s method for polychromatic staining of epoxy-embedded semithin sections. Histochem Cell Biol. 2020;153(2):121-7.
• Grimley PM. A tribasic stain for thin sections of plastic-embedded, OsO4-fixed tissues. Stain Technol. 1964;39(4):229-33.
• Gevondian TA, Avtandilov GG. Method for the finding and morphometry of cardiomyocyte mitochondria in semithin sections. Biull Eksp Biol Med. 1982;93(5):111-3. Russian.