At the moment, the staining of the milk (immunoglobulines?) and the E. coli is too strong to evaluate the epithelial cells.
Hello,
you have different possibilities.
Increase wash steps to remove reagents there remain in tissue
Increase H2O2 Block
Use a Protein block again unspecify reactions
Maybe it would be helpful to post your protocol
If it is actually milk, it may be full of biotin, that gives the unwanted background by binding streptavidin-peroxidase-konjugat. Look for biotin-block.
Thank you both! The protocol:
1. wash in TBS with 0.1% Tween
2. protein block serum-free solution
3. TBS and primary antibody (anti-CHI3L1/YKL-40 Antibody IHC-Plus (LS-B12326))
4. wash in TBS with 0.1% Tween; then
TBS and secundary antibody (Rabbit anti-mouse biotine)
5. wash and tertiair antibody (labelled polymer-HRP anti-rabbit)
6. wash and colour with DAB
7. rinse, wash and colour with hematoxyline, wash again.
If the previous suggestions are not efficient enough, I would strongly suggest to use an alternate reporting system such as alkaline phosphatase-based systems, for instance.
In clinical histology we routinly stain human mammary glands without problems. Is this an active lactating mamma? which species?
These are active lactating mammae of mouse. At this moment, the milk is stained a lot more than the epithelial cells.
You use a primary-antibody raised in mouse on mouse-tissue? You use a secondary-antibody raised in rabbit that binds to mouse-immunoglobins? I think that could be the cause of your problem. I hope you can find a primary from a different species.
i completely agree with Gudrun. Use a rabbit polyclonal primary Ab, which has been tested for IHC and works in Hu Ms and Rt, such as the one from United States Biological (see the link) an then the labelled polymer-HRP anti-rabbit. This is the most straightforward.
It exists tricks when one need to deal with Ab raised from a species on the same species, but we do not need to worry here in the first instance ! ;-)
Good luck with your IHC staining !
http://www.biocompare.com/9776-Antibodies/7414234-CHI3L1-Chitinase-3-like-1-ASRT7-DKFZp686N19119-FLJ38139-GP39-HC-gp39-HCGP-3P-YKL40-YYL-40-Antibody-CHI3L1-Chitinase-3-like-1-ASRT7-DKFZp686N19119-FLJ38139-GP39-HC-gp39-HCGP-3P-YKL40-YYL-40-Pab-Rb-xHu/?ncatid=9776&ppim=7414234_0_0
Thank you all for your reply! Mrs. Lang, would it be possible to share your protocol please? Thank you in advance!
the first antibody we use is a rabbit anti-mouse (LS-B12326). I don't understand why this would be a problem?
You wrote your secondary is a rabbit-anti-mouse-biotin. This implicates, that the primary is a mouse-antibody.
Your secondary ab will bind to any mouse-immunoglobin in the section.
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