Methods and Techniques Discussion > Immunohistochemistry (IHC)

Optimal protocol for staining of human liver?

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NBjorkstrom:
Hi all,

A student of mine have for the last month or so been trying to optimize a protocol for IHC-staining of snap-frozen human liver tissue. However, she's having problems with getting a good peroxidase block as well as with unspecific background staining. She's worked out two protocols that gives her decent, however not optimal, stainings. They are as follow:

1. Fix in 4% PFA
2. mAb block with 2% FCS in TBS-Saponin buffer
3. Peroxidase block either with 0.3% H2O2 in methanol or 2% H2O2 in TBS-Saponin
4. Block of endogenous biotin with the Avidin/biotin blocking kit from Vector
5. Stain with primary mAb o.n.
6. Development with the Vectastain ABC method and hematoxylin counterstaining

She gets good positive staining, however also significant levels of background.

Does anyone have experience with IHC-staining of human snapfrozen liver tissue? Or with peroxidase block of liver tissue? There seems to be a very high peroxidase activity in the liver, even after 15min x 2 incubation with 2% H2O2 in TBS-Saponin, the reaction goes on on the slides.

Any comments or thoughts are much appreciated!

CanuckPhD:
I have found that in fresh frozen liver it is very difficult to quench all of the peroxidases. It is one of the few times that I prefer to use fluorescent staining if I want to make a "pretty" picture. However, if you need to use IHC quench as much as you can (2 x 15 minutes in PBS as you state is good) and perform IgG controls to demonstrate what a negative stain looks like. This will at least show what the background is and what is positive staining.

For the biotin problem this is also a well known artefact when working with liver. I usually use the biotin free staining kits for liver (plus kidney and gut) to avoid this issue.

wallywoo:
We use DAB-Ni for 10 minutes to reduce Peroxidase autoflorescence in florescent staining.

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