Hi Viviane, as far as I know, when performing immunostainings in paraffin-embedded sections you need to add a step after the rehydratation steps called "antigen retrieval" (for instance with citric acid) in order to make the tissue available to the antibody and eventhough, there are antibodies that are unsuitable for paraffin-embedding. So, make sure you add this step and if the primary antibody you are using has been previously used on paraffin.

I don't know how much background you have, but it is a normal issue using paraffin. You have to make sure that your signal is easily visible yet the paraffin background.

I have a similar problem, looking for paraffin suitable antibodies!

Hi Mireia, thank you for your answer. I forgot to mentioned it, but i actually do the antigen retrieval step in my protocol (20 min in boiling 10 mM sodium citrate pH6.0). 

I'll definitely going to try different antibodies!

Hi Viviane,

unspecific backgroundstaining could have different reasons. You have to find out weather it depends on the primary antibody or on the secondary ab or on your detection system (Streptavidin, ABC, PAP-Technique). Therefore you should use a negative control. That means  you should react one tissue slide  withoute primary ab but with the complete protocol. Is your negative control negative, then you have to play with different delutions and with and without antigen retrievel (delution raw is highly recommanded to optimize the result). Do you get a posive result in your negative control then you have to think about different reasens why  sec. ab and/or detections system coul bind unspecifically.

1. if you are staining your Immuno with DAB you have to block the endogenous peroxidase (You can do that during    deparaffinating  the sections: Xylene 2 x 10 min, Isopropanol 10 min, H2O2 1:100 in 100% Ethenaol  in the fridge for 20 min.; continue wtih descending Ethanol up to destilled water; use fresh solutions). 

2.your secondary ab could bind unspecific on big molecules. Here you should block with BSA (try lower concentrations like 1% in 0,2% Triton-X in PBS or TBS) or better with normal serum (use the serum from the host animal of your secondary ab: Goat-Anti Mouse use normal Goat serum; I prefere the normal sera from Vector Labs).

3. If you are working with Strepavidin or ABC you have to block the endogenous biotin (the companies who sale the detection systems sale blocking kits as well; Abcam, Vector Labs etc.).

You can counterstain your immuno with Haematoxaline after you have rinsed the sections  in  the last buffer. After the countertatin dehydrate the ssections in ascending alcohols, clear them in  Xylene and mount them for example in DPX..

Eosin is a dye to stain  cytoplsm and not suitabal for counterstaining  immuno.

Hope, this information will help you to get better results. Good luck and best regards.

Hi Hussein and Ute,

Thank you for your answers.

I'm actually fixing my muscle in 10% formalin ON before paraffin embedded. Following deparaffinization and antigen retrieval, i'm blocking with MOM and then with BSA. I'll definitely try using the goat serum.

I'm trying to do both DAB and IF staining and both methods show a lot of background. I'm presently trying different dilution for the primary and secondary antibodies. 

Hi Viviane,

I have done many IHC paraffin tissue. Hers is the simple solution could solve your background problem:

1. 15 minutes room temperature incubation with 0.3% H2O2 in water  to remove the endogenous peroxidase. 

2. Block with serum different from your tissue specie, purchase from Vertor lab for 30 minutes at room temperature

3, Incubate (try 1:50 dilution) 2 hours at room temperature with your antibody-HRP conjugate if you cam find it commercially availble.

4. Wash 3X 10 minutes with 1X PBS tween20 (0.05%)

5. Develop with Immpact DAB from vector lab, monitor the signal under microscope until you see your specific antibody signal, it is very quick 1-2 minute, quickly wash with tap water and counter stain with Hematoxylin.

Hpoe that could solve your problem. Good luck

Hi Khuong,thank you for your advices! 

Hi Viviane

Did you find a working protocol?

I am currently facing the same problem you did!

Can you help me out with protocol you used for PAX7 on muscle tissue

Hi Ashis,

I tried different protocoles, but didn't obtained any good results. I now stop trying to optimize the staining of Pax7 on paraffin muscle slides and switch to OCT frozen muscle tissus.

Hi Viviane,

Thank you for the reply!

I was wondering whether you observed PAX7 staining in the cytoplasm instead of nucleus, when you do immunocytochemistry.