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Practical 3 and 4Immunohistochemistry
Course Immunology and Medical genetics
2nd year, Medicine, 2017/18
Immunohistochemistry
Protocol1. Fixation of tissue (PFA, frozen sections)
2. Deparaffinization/hydratation of tissue sections
3. Antigen Unmasking/Retrieval • 10 mM Na-citrate pH 6,0 or Tris/EDTA pH 9.0
4. Quench endogenous peroxidase• 3% H2O2 in H20
5. Blocking • 1% BSA/PBS 1-2 h RT
6. Primary antibody• diluted primary antibody in 1% BSA/PBS O/N +4°C
7. Secondary antibody• diluted secondary antibody in 1% BSA/PBS 1 h RT
8. Peroxidase substrate (DAB - 3,3'-diaminobenzidine)
9. Counterstaining (haematoxylin)
10. Re-hydration of tissue sections
http://www.youtube.com/watch?v=HdBgTAAi3rU
Deparaffinization/hydratation
• wash in xylene 2x 5 min
• wash in 100% EtOH 2x 5 min
• wash in 95% EtOH 2x 3 min
• wash in 70% EtOH 1x 3 min
• wash in PBS 1x 5 min
De-hydratation
• counterstaining – haematoxylin10 sec
• wash in hot water
• wash in 70% EtOH 1x 3 min
• wash in 95% EtOH 1x 3 min
• wash in 100% EtOH 1x 5 min
• wash in xylene 2x 5 min
Ki67 staining
Immunoblotting methods
• Methods based on the principles of antibody/antigen specific interaction
Use of immunoblotting methods
• Protein detection separated by gel electrophoresis and transferred on membranes (Westernblotting)
• The localization of antigens in tissue section (Immunohistochemistry)
• Antigen detection on cell surface (Flow cytometry)
• Quantitative determination of antigens in solution (RIA, EIA, ELISA)
SDS-PAGE (Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis
Proteinmarker
Proteins have the same charge-to-mass ratio Amino acid: SDS = 2:1
Protein visualization in gel
• Coomassie brilliant blue
• Silver staining
BSA 20 µg 10 µg 5 µg 2 µg
Proteinmarker
Protein transfer
• Nitrocellulose membrane
• PVDF (poliviniliden difluorid) membrane
1. PonceauS – visualization of proteins on membranes, reversible dye
2. BLOCKING in 5% BSA (Bovine serum albumin) TBS buffer
3. PRIMARY ANTIBODY incubation O/N
4. WASHING in TBS Tween-20 buffer (Tris Buffered Saline)
5. SECONDARY ANTIBODY incubation
6. WASHING in TBS Tween-20 buffer (Tris Buffered Saline)
7. Incubation with luminescent substrates for horseradish peroxidase (HRP)
8. VISUALIZATION of light signal on film
WESTERN BLOTPonceauS Immunoblotting
Antibodies used for immunodetection
• Primary antibodies• Polyclonal antibodies – antibodies produced
in animals (rabbit, mouse) immunized with purified protein and isolated from animal serum
• Monoclonal antibodies - hybridomas
• Secondary antibodies – produced in bigger animals (goat, sheep, donkey), recognize conserved region of primary antibody, labeled(e.g. goat anti-rabbit antibody)
www.abcam.com
Production of monoclonal antibodies
http://www.sumanasinc.com/webcontent/animations/content/monoclonalantibodies.html
Production of polyclonal antibodies
Antibody labeling• Fluorescent probes, fluorophores (fluorescein, rhodamine, fikoeritrin)• Radioactive isotopes (I125, H3)• Enzyme conjugation – horseradish peroxidase (HRP), Alkaline phosphates
Detection• Immunofluorescence (fluorescent product)• Colorimetric (densitometry - colored and insoluble product, photometry –colored
product soluble in solution)• Luminescence (bioluminescence, chemiluminescence)
Bioluminescence: Luciferin oxidation by luciferase, enzyme from the firefly Chemiluminescence: Luminol oxidation in presenceof hydrogen peroxide, product 3-aminophthalate is inan excited electronic state, which then decays into anelectronic ground state, result is emission of light detected on X-ray film
Immunofluoresence - localization of protiens in cell
http://www.sigmaaldrich.com/Blue – DAPI staining of DNA
Ki 67 staining – WB, IHC, IF, flow cytometry, ELISA
Western blot
Immunohistochemistry
Immunofluoresence
flow cytometry
ELISA
SDS PAGE: http://www.youtube.com/watch?v=toPpdoBYPWoWestern blot: http://www.youtube.com/watch?v=pRIn3p7x3V8Immunofluorescence: http://www.youtube.com/watch?v=pteO6FRWo3gImmunohistochemistry: http://www.youtube.com/watch?v=HdBgTAAi3rU