imunohistokimia-pengantar s1
TRANSCRIPT
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DWI WINARNIDepartemen Biologi
Fakultas Sains dan Teknologi
Universitas Airlangga Surabaya
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A. Definisi
visualisasi bagian jaringan atau sel melalui deteksi interaksi antibodi-antigen spesifik, dimana antibodi dilabel dengan
label atau marker
- Pewarna fluorensens
- koloid logam
- hapten
- marker radioaktif
- enzim
Beragam mulai wujud sel atau irisan beku hingga sel atau jaringan dalam blok parafin atau resin
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dalam immunostaining, antibodi digunakan untuk membentuk link antigen yang ada pada sel atau jaringan secara spesifik pada pewarna (reaksi yang menghasilkan warna) sehingga keberadaan antigen tersebut teramati di bawah mikroskop
immunohistochemistry immunocytochemistry
antigen dalam jaringan Antigen dalam kultur sel
Cryostat section
Memerlukan fiksasi
paraffin section resin section
Tanpa fiksasi
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History of immunohistochemistry
1942 Coons, Creech, Jones, Berliner
Developed indirect immunofluorescence method
1959 Singer Developed electron-dense molecular conjugate (ferritin)
1965 Sternberger Developed electron-opaque heavy metal technique (uranium)
1966 Graham & Karnovsky
Developed enzyme tagging method (horseradish peroxidase
1967 Nakane & Pierce Developed enzyme-labelled antibody technique (immunoperoxidase)
1970 Sternberger Developed unlabelled antibody method (PAP method)
1971 Faulk & Taylor Developed another electron-opaque heavy metal technique (colloidal gold)
1974 Heitzman & Richards
Developed another unlabelled antibody method (ABC technique)
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Prinsip imunohistokimia
• Reaksi antigen-antibodi untuk lokalisasi antigen
• Labeling dengan agen pewarna/fluoresens atau electron-opaque substance (ultrastructural tags) untuk memvisualisasi reaksi Ag-Ab
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B. TAHAP-TAHAP DALAM IMUNOHISTOKIMIA
B.1. ANTIGEN RETRIEVAL
Antigen retrieval/unmasking dengan melakukan pretreatment spesimen
Meningkatkan reaktivitas antigen dalam jaringan
Mutlak dilakukan untuk jaringan yang difiksasi formalin karena fiksatif mengadakan cross link inter dan intramolekular dengan protein struktural tertentu yang dapat mengakibatkan masking antigen
Pretreatment : digesti menggunakan enzim proteolitik, pemanasan, iradiasi microwave, autoclaving atau pressure cooking
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B.1. 1. Digesti menggunakan enzim proteolitik
B.1. ANTIGEN RETRIEVAL
Pronase 0,05% w/vProteinase-Ktrypsin 0,05% w/vpepsin 0,05%
!!!!!!!! Tidak semua antigen memerlukan digesti proteolitik karena enzim proteolitik dapat menghilangkan reaktivitas antigen tertentu, menimbulkan antigenic sites palsu , atau mengubah sifat serta merusak antigen
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B.2. IMMUNOLABELING
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Visualized antigen-antibody reaction by adding SUBSTRATE & CHROMOGENS
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Sampel : pankreas rat (fiksatif formalin/PFA)Antigen retrieval : -Blocking : BSA 2%, 2 jam 25oCPrimary antibody : mouse-anti –rat insulinSecondary Ab : goat-anti-mouse HRP conjugationChromogen : DAB
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.substance P merupakan primary afferent derived neuropeptide .jumlah reseptor dan kadar substance P meningkat
pada kondisi pain persistent (ex: inflamasi kronis)
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CGRP (Calcitonin gene related peptide ) is one of the most abundant peptides produced in both peripheral and central neurons. It is the most potent peptide vasodilator and can function in the transmission of pain. In the trigeminal vascular system the cell bodies on the trigeminal ganglion are the main source of CGRP. CGRP is thought to play a role in cardiovascular homeostasis andnociception.
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APOPTOTIC CELLS DETECTION