Immunohistochemistry

Frequently Asked Questions (FAQ)

 

Dr. Giorgio Cattoretti
Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA

 

 

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IHC FAQ Home

Frozen or paraffin sections?

Answer:

 

Some epitopes do not survive fixation and embedding.

From the same frozen block you can get DNA, RNA, free nuclei for FISH or cell cycle analysis, protein and a stained section on serial sectioning.
 

From a paraffin block you can get DNA and RNA for PCR amplification (extensive crosslinking prevents extraction of long nucleotide stretches), free nuclei for ploidy and cell cycle analysis, cells for flow cytometry and several section on serial sectioning.

Fixation and embedding prevents degradation and physical relocation of antigen (protein, DNA, RNA) by crosslinking.
 

Morphology on frozen is poor. Paraffin sections allow unlimited almost identical serial sections.
 

Cell and tissue shrinkage, associated with fixation and embedding, results in higher antigen density on the section.
 

Nasty bugs are destroyed by fixation
 

Most of the specimens stained in a routine immunohistochemistry lab in Surgical Pathology are formalin-fixed, paraffin embedded tissues. Fixation and embedding cause antigen masking, but also better retention of labile proteins, nucleic acids and small peptides. In order to overcome the drawback of antigen loss, enzymatic- or heat- mediated antigen retrieval is used.

 

The methods detailed here, mostly tailored for paraffin sections, can be applied to frozen sections, cytospins, smears.

 
On such material, you may have to deal with significant presence of

endogenous enzymes (AP, AcPh, peroxidases)

endogenous biotin

Fc receptors

loss of cellular antigens and morphologic detail

diffusion artifact

You may not use antigen retrieval on frozen sections: there is no fixation and embedding-associated antigen masking. You may try heath-mediated quenching of endogenous phosphatases, but on crosslinking-fixed specimens only.