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Immunohistochemical Analysis of Paraffin-Embedded Tissue
Turkey (TurkMSIC) - Rize University, Rize
Department of Histology and Embryology, Recep Tayyip Erdogan University, Faculty of Medicine, Fener Mh., 53100 Rize, Turkey
Assoc. Prof. Tolga Mercantepe
Assoc. Prof. Levent Tumkaya Assist. Prof. Tolga Mercantepe
Type of Research Project
- Basic science
What is the background of the project?
Immunohistochemistry (IHC) is used to understand the distribution and localization of proteins in tissues which detects specific antigens using an appropriate antibody labeling strategy. Immunohistochemistry (IHC) is the gold standard methodology for in-situ protein expression analysis in tissue samples. For this purpose, samples are collected, fixed and antigenicity of target epitopes, and then sectioned. A variety of antibody staining schemes can produce informative IHC images.The Turkish Society of Histology and Embryology has been founded in 1990 with collaboration of both veterinary and medical professionals.Turkey has established IHC units and embryologists who work on two major fields: identification of genetic abnormalities in embryological tissue samples during or after pregnancy, and research related to natural developmental processes of the fetus. Our unit works with 6 histology/embryology specialists and has access to IHC labs and molecular imaging techniques.
What is the aim of the project?
Investigating immunohistochemical levels of various antibody in different tissue samples.
What techniques and methods are used?
We used a caspase-3 (rabbit polyclonal to caspase-3, ab4051, Abcam, UK) immunohistochemistry kit as a primary antibody to mark apoptotic cells. Sections 3-4 μm taken from the paraffin blocks using a microtome (Leica RM2125RT, Germany) were placed onto positively charged slides. The sections obtained were then kept for 10 min in 3% H2O2 solution. The sections were next washed in phosphate buffer solution (PBS) and incubated with citrate buffer solution for 5 min, and were then kept for 15 min in secondary blocking solution. They were then incubated with primary antibody (Caspase-3, Rabbit polyclonal to Caspase-3, 1/100, ab4051, Abcam, UK) and secondary antibody (EXPOSE Rabbit specific HRP/DAB (HRP: horseradish peroxidase, DAB: 3, 3-Diaminobenzidine) detection IHC Kit, ab80437, Abcam, UK) for 60 min. Finally, sections stained with Harris hematoxylin and diaminobenzidine chromogen solution were covered with entellan (Superior Mansfield, Germany).
What is the role of the student?
- The student will mainly observe
What are the tasks expected to be accomplished by the student?
The student will observe or be involved under supervision in the following tasks: 1- Marking of apopitotic cells with Caspase 3 2- Using the Leica RM2125RT microtome to prepare the paraffin slides used for imaging 3- Preparing the microslides using a 3-step fixation procedure; 10mins at 3% H2O2 then wash with phosphate buffer then incubate with citrate buffer for 5 mins 4- Observing the use of the EXPOSE Rabbit specific HRP/DAB (HRP: horseradish peroxidase, DAB: 3, 3-Diaminobenzidine) detection IHC Kit 5- Occasionally examining the embryonic tissue slides prepared in the IHC lab
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Immunohistochemical (IHC) Analysis Procedure –Assit. Prof. Tolga Mercantepe (courses, seminars)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
No specific skills are required.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students
- Mercantepe; Tolga; Tümkaya; L.; Çeliker; F.B.; Topal Suzan; Z.; Çinar; S.; Akyildiz; K.; Mercantepe; F.; Yilmaz; A.; 2018. Effects of gadolinium-based MRI contrast agents on liver tissue. J. Magn. Reson. Imaging 1–8. https://doi.org/10.1002/jmri.26031
- Mercantepe; T.; Unal; D.; Tümkaya; L.; Yazici; Z.A.; 2018. Protective effects of amifostine; curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats. Exp. Ther. Med. 15. https://doi.org/10.3892/etm.2018.5819
- Topcu; A.; Mercantepe; F.; Rakici; S.; Tumkaya; L.; Uydu; H.A.; Mercantepe; T.; 2019. An investigation of the effects of N-acetylcysteine on radiotherapy-induced testicular injury in rats. Naunyn. Schmiedebergs. Arch. Pharmacol. https://doi.org/10.1007/s00210-018-1581-6
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