Principle of Immunohistochemistry

Immunohistochemical staining• light microscope•Imaged by color development•Cryosection or paraffin-embedded section

Immunofluorescent staining• immunofluorescent microscope•Imaged by flourochrome excitation•Cryosection or cultured cells

Glutaraldehyde Carnoy’s solution

1day 336 days 1day 336 days

Glutaraldehyde: 15.5ml 25% GA+12.5ml phosphate buffer +dH20 to 100mlCarnoy’s solution: 60ml aboslute ETOH+30ml CHCl4+ 10ml 100% Acetic acid

J Histochem. Cytochem. 2006 54:371-74

Effect of fixation on tissues4% phosphate buffered formalin

FGA 28days

TC11 56days

TC11 84 days

Mt DNA

Effect of fixation on tissues

Specificity control for IHC

Controls for the specificity of an antibody are most important for the correct interpretation of its localization in cells and tissues

• Absorption control

antibody mixed with the protein or peptide used to generate the antibody, with the objective of eliminating the binding of the antibody to the protein in the tissue

Drawback: determines only the specificity of the antibody for the incubating protein or peptide and does not prove the specificity of the antibody for the protein in the tissue

Specificity control for IHC

Controls for the specificity of an antibody are most important for the correct interpretation of its localization in cells and tissues

• Specificity of the antibody1. determined by showing specific reaction with the peptide or protein used for immunization with highly sensitive methods other than IHC

→ immunoblot

→immunoprecipitation

2. details of antibody generationsequence of amino acids used, species from which the immunogen obtained, isotype of immunoglobulin generated

Specificity control for IHC

Controls for the specificity of an antibody are most important for the correct interpretation of its localization in cells and tissues

• Specificity of the IHC method

1. labeling is specifically due to the primary antibody

→ normal serum control

→ a known unreactive antibody control → nonspecific binding control

2. confirmation of Ab specificity with tissues (cells) containing the protein of interest

• Definition: predominantly (originally) defined as a high-temperature heating method to recover the antigenicity of tissue section that had been masked by formalin fixation

• Purpose: amplification in pre-detection phase by reduction of the detection thresholds of immunostaining (increasing sensitivity) and retrieval of some antigens

• Mechanism of action

1. release calcium from the cage-like calcium complex formation with proteins in formalin-fixed tissue

2. lossening of breaking of the crosslinkage induced by formalin fixation

• Applicationparaffin-embedded section, plastic embedded tissues, celloidin-embedded tissues, non-embedded tissue slices, cell smear preparations, ISH, Tunnel, FISH

• Caution1. not all antigen modified by formalin can be restroed using conventional AR protocols2. high temperature may induce a negative result of IHC staining3. pH value of the AR solution

Microwave Vacuum Histoprocessor

AR for FISH

Principle of Immunohistochemistry

Immunohistochemical staining• light microscope•Imaged by color development•Cryosection or paraffin-embedded section

Immunofluorescent staining• immunofluorescent microscope•Imaged by flourochrome excitation•Cryosection or cultured cells

Light Microscope

1. Bright-field microscope2. Dark-field microscope It is different form the bright field one in the condenser lens function and is useful for viewing particles far below the limits of bright light resolution by darkening the background.3. Phase contrast microscope With a specifically designed condenser lens, it can be used to examine living cells and unstained tissue by utilizing differences in the diffraction index of cell or tissue component. 4. Differential interference microscope Using Normaski optics, it is useful for surface examination of the object.5. Fluorescence microscope It is equipped with an ultraviolet light source to detect tissue autofluorescence or fluorescence-labeled components of the tissue.6. Confocal scanning microscope It is designed to combine light microscope components, fitted with fluorescence equipment and a scanning system, with a computer system to image the specimen in very thin optical sections by subtracting out-of-focus regions, and even create an 3-D image through stacking a series of images at varying depths.7. Ultraviolet microscope With UV light source, images are viewed through photographic records rather than an ocular.8. Polarizing microscope It is useful for detecting crystalline substances and well-ordered fibrous molecules due to the result of birefringence.

Phase contrast microscope

Differential interference microscope

Principle of Immunofluorescent microscopic staining

Double immunofluorescence of Pinin and cytokeratin

( Ouyang and Sugrue, JCB 1996)

Triple immunofluorescent staining of mitotic spindle