Fourth complex is formed by incubation with a secondary,

Fourth technique is Immunohistochemistry which usesantibodies to test for certain antigens (markers) in a sample of tissue. Theantibodies are linked to an enzyme or a fluorescent dye. When the antibodiesbind to the antigen in the tissue sample, the enzyme or dye will be activated,and the antigen can be seen under a microscope National Cancer Institute, 2018.                  Figure11: Immunohistochemistry Staining Equipment BioGenex, 2017            Principle for this technique isthat it is developed from the antigen-antibody binding reaction that visualizesdistribution and localization of specific antigen or cellular components inseparated tissues, or tissue sections.

Fluorescent or chromogenicsignal for protein detection can be produce by direct or indirect detectionmethods. Direct detection is when the primary antibody specific for the targetmolecule is directly labeled while indirect detection uses an unconjugatedprimary antibody Novus Biological, 2018.  Majorcomponents in immunohistochemistry includes primary antibody binds to specificantigen, the antibody-antigen complex is formed by incubation with a secondary,enzyme-conjugated, antibody and lastly, with the presence of substrate andchromogen, the enzyme catalyzes to generate colored deposits at the sites ofantibody-antigen binding Novus Biological, 2018.

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                Figure 12: Immunohistochemistry Process ImmunohistochemistryUS, 2018.     The Advantages forfluorescent detection include easy multiplexing, better target co-localization,higher dynamic range and fewer steps. For chromogenic detection, it has greatersensitivity and longer lasting signal NovusBiological, 2018 For applications, this technique involves in Prognostic markers in cancer. Physicians diagnose cancer asbenign or malignant by using specific tumor markers. This technique can alsodetermine the grade and stage of a tumor, and identify the cell type and originof a metastasis to find the site of the primary tumor Jeyapradha, D., 2012. For genetics, it determines the function of specific gene products in fundamental biologicalprocesses such as apoptosis and development. A custom made monoclonal antibodywas used against p53 homologue of the pro-apoptotic pathways of p53 wasidentified Jeyapradha, D.

, 2012. Lastly is Tumors ofuncertain histogenesis which diagnose tumors of uncertainorigin, primary as well as metastatic from unknown primary tumor. A panel ofantibodies is chosen to resolve such diagnostic problem cases. Jeyapradha, D., 2012.              Last technique is Tissue in Situ Hybridization.

Before this, researches are focus on the identification ofspecific DNA targets using RNA or DNA labeled with radioisotopes.  Now, the use of the in situ hybridization(ISH) technique is popular. The use of interphase FISH in routinely formalinfixed paraffin-embedded (FFPE) tissue to study cytogenetic abnormalities hasbecome common Mary, H.

, 2009.  Types of Probes used are DNA Probes which detect DNA targets. They are generated by the cloning andamplification of specific sequences of DNA or cDNA Mary, H., 2009.

 Second is RNA Probes which detect RNA in tissue sections. It is known asRiboprobes which generated by in vitro transcription from plasmids containingthe sequence of interest Mary, H.,2009.

Next is Oligodeoxynucleotideprobes which are short sequences of DNA generatedon an automated DNA synthesizer Mary,H., 2009. Lastlyis PNA (polypeptide nucleic acid) Probes which is a DNA analogue thatforms very stable duplexes with complementary DNA or RNA sequences Mary, H., 2009.  Figure 13: FISH Digital Scanner Leica Biosystem, 2018   The principle for this technique is that it identifies where mRNAs are present in a fixed tissue samples and detect nucleic acid sequences in solid neoplastic and infectiousconditions.

Furthermore, it also detects microRNA in cytological preparationsand tissue sections recently Mary, H.,2009.             Figure14: FISH Techniques Steps VeterianKey, 2017.

            Figure15: FISH using oligonucleotide probes VeterianKey, 2017. The advantages is detect protein and  mRNA of interest or cells phenotype, detectmore than one nucleic acid sequences using different labeling methods, enablesmaximum use of tissue that is difficult to obtain (embryos and biopsies) andperform hundreds of different hybridizations on the same tissue Ellen, J.,2014. Applications for this technique are in Genomic abnormalities in cancer which provides information on thelocations of important cancer genes and can have clinical use in diagnosis andcancer classification. It alsoapplied for monitoring the progression of tumors Bassem,A., 2006.

It also involves in Submicroscopic aberrations. Prader–Willi syndrome (PWS) is a paternal structuralabnormality involving 15q11-13, while a maternal aberration in the same regioncauses Angelman syndrome. These small aberrations can detected using array CGH Hassan, M., 2016. Lastlyis in prenatal genetic diagnosis which uses array CGH inpreimplantation genetic screening is becoming an increasingly popular. It hasthe potential to detect CNVs and aneuploidy in eggs, sperm or embryos which maycontribute to failure successfully implant or miscarriage Evangelidou, P.

, 2013.                        Conclusion As a conclusion, advanced techniquesthat are available right now are very advantageous and can replace the oldtechnique. However, there are also drawbacks in the advanced techniques.

Manufacturers and researches can make a team to improve the advanced techniquesso that it can make improve the diagnosing quality in future to benefits thepatients. Furthermore, scientist can make anew technique that combines the old technique and advanced technique to producea new technique that benefits in diagnosis and research because the oldtechniques has many advantages also.