Serap ŞİRVANCI, Özlem Tuğçe ÇİLİNGİR KAYA, Cynthia MOORE, Charles MESHUL

Nörogenez Belirteci Olan Doublecortin Proteininin Elektron Mikroskopik Düzeyde Gösterilmesi

Amaç: Yetişkin beyninde yeni oluşan nöronların gösterilmesi nörogenez sürecinin aydınlatılması açısından önem taşımaktadır. Çalışmamızda hipokampusun dentat girus (DG) bölgesinde, yeni oluşan nöron belirteci olan doublecortin (DCX) proteini için uygun gömme öncesi mikrodalga işaretleme metodu oluşturmayı amaçladık. Yöntemler: 10 haftalık C57BJ/6J erkek farelerden perfüzyon fiksasyonu ile beyin dokuları elde edildi. Vibratom ile beyin dokularından alınan kesitler, gömme öncesi mikrodalga yöntemi ile anti-DCX antikoru ile işaretlendi. 3,3'-diaminobenzidin (DAB) ile boyanan dokular, mikrodalga yardımı ile elektron mikroskopik analizler için hazırlandı. Epon bloklar üzerindeki dokulardan ince kesitler alınarak elektron mikroskobunda incelendi ve morfolojik analiz için fotoğraflandı. Bulgular: Mikrodalga yöntemi kullanılarak yapılan gömme öncesi DCX işaretlemesi hem ışık hem de elektron mikroskopik (EM) düzeyde spesifik olarak gösterildi. DCX pozitif hücrelerin subgranüler zon (SGZ) ve granüler tabakada yerleşim gösterdikleri izlendi. Elektron mikroskopik incelemelerde DCX immünreaktivitesinin akson, dendrit ve hücre gövdeleri üzerinde pozitif olduğu gözlendi. Sonuç: Çalışmamızda, mikrodalga kullanılarak yapılan gömme öncesi immün-elektron yöntemi ile DG bölgesinde DCX varlığının belirlenebildiğini gösterdik. Çalışmamız DCX immünreaktif hücreleri, gömme öncesi DAB işaretlemesi ile elektron mikroskopik düzeyde göstererek, nörogenez sürecinin araştırılacağı ileri çalışmalar için temel oluşturmaktadır.

Anahtar Kelimeler:

Nörogenez, doublecortin, gömme öncesi mikrodalga tekniği, immün-elektron mikroskopi

Demonstration of Doublecortin Protein, a Neurogenesis Marker, at the Electron Microscopic Level

Objective: Demonstration of newly born neurons in adult brains is an important issue in terms of elucidating neurogenesis. In our study, we aimed to develop an appropriate pre-embedding microwave labeling method for the newly born neuronal marker doublecortin (DCX) protein in the hippocampal dentate gyrus (DG) region. Methods: Brains were obtained from 10-week-old C57BJ/6J male mice by perfusion fixation. Vibratome sections from brain tissues were labeled with an anti-DCX antibody using a pre-embedding microwave method. Sections stained with 3.3'-diaminobenzidine (DAB) were prepared for electron microscopic (EM) analyses using a microwave. After embedding in Epon, thin sections were obtained, observed under an electron microscope, and photographed for morphological assessments. Results: DCX labeling performed using the pre-embedding microwave method was specifically demonstrated at both light and electron microscopic levels. DCX-positive cells were localized at the subgranular zone and granular layer. Electron microscopic observations showed that DCX immunoreactivity was positive at the axons, dendrites, and somata. Conclusion: We demonstrated that the existence of DCX can be determined using the pre-embedding microwave labeling as an immunoelectron method in the DG region. Our study provides a basis for further studies on neurogenesis aiming to show DCX-immunoreactive cells using the pre-embedding DAB labeling method at the electron microscopic level.

Keywords:

Neurogenesis, doublecortin, pre-embedding microwave technique, immunoelectron microscopy,

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