Immunostaining

Methods Of Immunostaining

1. IMMUNOSTAINING SOUVIK BISWAS M.PHARM (PHARMACOLOGY), 1 ST YEAR (2 ND SEM) ROLL NO. 27720216003 REGISTRATION NO.162772310018 UNDER THE GUIDANCE OF DR. SANDIPAN DASGUPTA NSHM KNOWLEDGE CAMPUS, KOLKATA – GROUP OF INSTITUTION

2. Antigens Antigen is any substance (such as an immunogen or a hapten) foreign to the body that evokes an immune response either alone or after forming a complex with a larger molecule (such as a protein) and that is capable of binding with a product (such as an antibody or T cell) of the immune response.

3. Antibodies (immunoglobulins; Ig) It is a large number of proteins of high molecular weight that are produced normally by specialized B cells after stimulation by an antigen and act specifically against the antigen in an immune response and that typically consist of four subunits including two heavy chains and two light chains - called also  immunoglobulin.

4. WHY DO IMMUNOSTAINING? Confirm genotype-phenotype (e.g. KO, KI, levels or efficiency of expression, etc.) Where is a biomarker expressed? When is a biomarker expressed? Co-expression or co-localization. Cell type-specific biomarkers (e.g. CD4, CD8 T- cells , NK, Macrophages, Mast Cells , etc.)

5. Introduction and Definition Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. The key to immunohistochemistry is the specific antibody-mediated detection of a target antigen, known as immunostaining or immunodetection.

6. Immunostaining Method According to different biotins conjugated with antibodies, IHC staining methods can be classified as Immunofluorescence, Immunoenzymological staining Immunocolloidal gold technique According to different kinds of procedures, immunohistochemistry staining can be divided into subtypes of Direct staining (one-step staining) Indirect staining (two-step, three-step or multi-step staining) PAP staining method

7. different biotins conjugated with antibodies Immunofluorescence: It is the first immunohistochemical staining method. With antigen-antibody binding reaction, antigens are visualized by fluorescence dyes conjugated with antibodies and that is localized under fluorescence microscope. Immunoenzymological Staining: In here enzyme-labeled antibodies are used to bind with specific antigens in tissues samples or cultured cells and localized by light microscope.

8. Contd… Immunocolloidal Gold Technique: It is a kind of technique that uses colloidal gold as a marker. Immunocolloidal gold technique is suitable for single or multi-label detection under immune-electron microscope, and light microscope.

9. Different kinds of procedures Direct Staining: Incubate the sections with mixture of two primary antibodies which are respectively conjugated with two fluorescence dyes (e.g. FITC and TRITC) Or, successively incubate sections with two primary antibodies. Indirect Staining: In here the primary antibodies are without fluorescence dyes. Incubate sections with one kind of primary antibody and corresponding secondary antibody, then the other.

10. Contd… PAP Staining Method (peroxidase anti-peroxidase method): Incubate sections with corresponding secondary antibodies which are conjugated with two different enzymes (e.g. HRP, AKP), or anti-HRP (PAP complex), anti-AKP (APAAP complex).

11. Immunostaining Protocols

12. Immunohistochemistry Staining Tips Fresh fixed tissue Sufficient dehydration of tissues An intact, uniform and smooth sectioning Binder materials Sufficient deparaffinization A thorough inhibition of endogenous peroxidase

13. Applications Clinically, IHC is used in histopathology for the diagnosis of specific types of cancers based on molecular markers. In laboratory science, immunostaining can be used for a variety of applications based on investigating the presence or absence of a protein, its tissue distribution, its sub-cellular localization, and of changes in protein expression or degradation.

14. References Formation of the first cleavage spindle in nematode embryos by Albertson, D.GDev . Biol. 101, (1984). page no. 61–72. Immunohistochemistry: Methods Express, edited by Simon Renshaw, Scion Publishing Ltd, Bloxham, UK,  1 st edition (15 Dec. 2006), page no. 45-96 http://www.abcam.com/index.html?pageconfig=resource&rid=12615 (accessed on 16.05.2017) http://www.mdbioproducts.com/resources/protocols/immunohistochemistry (accessed on 17.05.2017) http ://www.immunohistochemistry.us/immunohistochemistry-staining.html (accessed on 18.05.2017)