[Histonet] RE: fixing immuno and antibody tissues

From:"Johnson, Teri"

Paul Webster gave a brilliant answer to George Cole's question about
fixation and its role in immunohistochemistry.  Patti Loykasek also gave
an excellent response, especially considering the IHC done on routine
surgical pathology specimens.  I totally agree with Patti in doing
everything you can to make sure you are not obtaining false positives
resulting from digestion or unmasking techniques.  Controls are critical
at this point!  Regardless of what is recommended on the antibody data
sheet, I will always work up a new antibody using no pretreatment, an
enzyme digestion (usually trypsin), and a HIER method using citrate
buffer pH 6.0 in the electric pressure cooker or waterbath. If none of
those conditions work, I'll try with different pretreatment regimens,
i.e. pepsin, protease, and/or high pH HIER.  Additionally, most of the
antibodies that are tested for use in IHC have been tested using
cryosections.  These tissues are generally not fixed, but snap frozen,
sectioned, allowed to air dry, and then fixed in acetone (or a mixture
of acetone/alcohol) before immunostaining.  Some labs will use
formalin-fixed, sucrose cryopreserved cryosections with good results,
but if formalin fixation renders the antigen inaccessible or not well
preserved, then this is no improvement from using formalin fixed,
paraffin embedded (FFPE) samples.
Luckily, for most clinical applications, many of the antibodies are well
characterized and optimized for use in FFPE samples.  Research
applications tend to be a little more challenging on this front.

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110

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