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

Abstract

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.

Keywords

• epon kesit
• özel boyamalar
• light green

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

Abstract

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.

Keywords

• epon sections
• special stainings
• light green

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