antigen retrieval technique

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Abhishek Singh
Abhishek Singh's picture
antigen retrieval technique

what is antigen retrieval technique.

The FFM
The FFM's picture
Basically by pretreating the

Basically by pretreating the antigens in your sample by one of many methods, you can improve the antigenicity by breaking crosslinking caused during tissue fixation and improve the detection of the antigen by the primary antibody
 
See  http://www.ihcworld.com/epitope_retrieval.htm  for a list of retrieval methods

Abhishek Singh
Abhishek Singh's picture
so if we have introduced the

so if we have introduced the antigen, so if there is less antigenicity in sample that can be enhanced but if there is no antigenicity then it will not.

The FFM
The FFM's picture
Not quite sure what you are

Not quite sure what you are saying there....but,
When you fix tissue for staining with antibodies, the cross-linking that occurs during fixation can lead to the masking of the epitopes that the antibody recognizes.  This leads to poor detection.
The antigen retrieval techniques are performed after fixation and before the application of primary antibody to reduce the crosslinking in the proteins enough that it improves the availability of the epitope to be detected by the antibody while the tissue itself remains fixed.
This basically is a way of increasing the real signal above the background non-specific staining.  If the antigen is not present in the tissue, you should not see an difference in staining of two serial tissue sections, the first which has been antigen retrieved and the second control section which has not.

clonegene
clonegene's picture
Pathology samples are  fixed

Pathology samples are  fixed in formaldehyde, embedded in paraffin blocks  (FFPE) , cut into sections, stained with dyes  and viewed under a microscope . This method  preserves cellular structure and reveals considerable detail. Such blocks are collected over many years and thus provide an invaluable clinical database for studies on  gene expression .  However, this method  crosslinks proteins  and thus  these sections do not  work well in standard  immunohistochemical analysis. To reactivate the reactivity with the primary antibody,  the sections are heated in a low pH buffer which removes the crosslinks. Some protocols recommend steam heat some recommend microwave. Many vendors provide a reactivation kit. However, it is important to note that not all antibodies can recognize  the reactivated uncrosslinked antigens.  This is particularly the case with monoclonal antibodies. However, some monoclonals are selected with precisely this purpose in mind and work very well on  FFPE sections. Importantly , immunohistochemical  analysis of FFPE tissues gives the highest resolution result and is much superior to staining on frozen sections.