Immunohistochemistry steps
Tissue sections
Antigen removal
Blocking endogenous enzymes
Primary antibody
Secondary antibody
Chromogen substrate
Counterstain
Mounting
Helps to prevent: elation , degradation and modification
Fixation
Preserves the positions of the tissue of the tissue architecture and cell morphology
Fixation
Preserves the secondary and tertiary structure to a possible extent
Fixation
Provides target for antibody molecules
Fixation
Proffered fixative
Formaldehyde
2-4 micron tissues are cut onto slides
Slide preparation
Charged slides provide adhesion to tissue sections
Slide preparation
The tissues are further adhered to the slides by baking at 60 degreees
Slide prepartion
Temp in slide preparation
60 degrees
Tissue is treated in a series of xylene and alocohol to remove paraffin
Deparaffinization
Sample is embedded in paraffin to manga in the natural shape and architecture of the sample during long term storage and sectioning for IHC
Fixation
Enables the partial reversal of formaldehyde induced confirmational change of antigens
antigen removal
Increases the accessibility of the ab to the ag
Antigen removal
Two methods in antigen removal
Heat
Enzyme digestion
Choice of antigen retrieval depends on the antigen to be demonstrated
Antigen removal
