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