Effect of Fixation on BrdU Immunohistochemistry

This adenocarcinoma tumor was bisected at necropsy. Half was fixed in 10% neutral buffered formalin for 24 hours and the other half was fixed in methacarn for 24 hours.

formalin hp 24t1.JPG (26220 bytes)

Formalin fixed

Formalin fixation produces excessive cross linking of proteins (formation of methylene cross bridges) which "mask" antigens. This "masking"  prevents the mouse anti-BrdU IgG from attaching to the incorporated BrdU. Staining is very weak to non-existent in this example

  formalin-ar hp 24t1.JPG (32759 bytes)

Formalin fixed / Heat Induced Epitope Retrieval (HIER)

The HIER technique was able to recover some, but not all of the BrdU epitopes. Staining is moderate in this example.

  methacarn hp 24t1.JPG (37119 bytes)

Methacarn fixed

Methacarn is a precipitant fixative and does not "mask" the antigen. Note the intense BrdU stain. Methacarn is an excellent fixative for most cell cycle related proteins  and cytoskeletal proteins. However, it is not recommended for demonstration of hormone receptors (ER/PR) and for PCR/in Situ applications.  

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