Hello everyone,
I am working with formalin-fixed, paraffin-embedded myocardium samples for staining of several markers. However, I cannot stain properly the nuclei with DAPI (image annexed). After deparafinization with xylene, we performed antigen retrieval with sodium acetate buffer. Nuclei staining was done with DAPI 2 ng/uL, 5 min. We also tried DAPI for more time and even Hoescht. We also used the TRUE VIEW Quenching Kit for background reduction and no differences were observed.
Does anyone have any ideas or suggestions for a correct staining?
Thank you in advance!
Hi, I don't have a specific suggestion as to why the DAPI isn't working, but have you tried a classical histochemical staining to check that the nuclei are able to be seen that way, such as a heamatoxylin/eosin staining? I can imagine there could be problem with the deparaffination steps, which could cause this problem for you, maybe you could provide some more details? We could imagine residual paraffin or xylene in the sample could impede proper staining of DNA. I hope this might be of some help, have a nice day!
Dear Jasper B.J. Kamphuis ,
Thank you for your help!
The protocol we use for deparaffination is the following:
1) 7 min in xylene, 2x;
2) 5 min in ethanol 100%, 2x;
3) 3 min in ethanol 90%, 2x;
4) 1 min in ethanol 70%;
5) 10 min in distilled water.
We also performed antigen retrieval with sodium acetate buffer (pH 6) for 20 min at 93ºC.
We are going to perform H&E staining, as suggested.
Thank you very much. Have a wonderful day.
Dear Gustav Stålhammar
,These are samples from old patients (> 75 years). In fact, it is curious that some samples work instead of others...
I don't have any information regarding the ischemia time. Maybe it is also an important factor for staining is not working as expected.
We are going to perform H&E staining as suggested.
I really appreciated your help. Have a wonderful day!
I agreed with previous suggestion, you should visualize the morphological structure of your samples using H&E to determine the localization of nucleus in your tissue samples. Best Wishes.
Hi! Same issue here... usually nuclei counterstaining with Hoesch worked always fine for my samples, except when I perform Heat Induced Antigen Retrieval. I loose completely the nuclei staining. Did you figure out what was wrong?
Protocol is the following:
- deparaffination with xylene (x3 - 3min each) and rehydratation (100% EtOH x3, 96% EtOH x3, 70% EtOH x1, distilled water - 3min each)
- HIER with either Tris-EDTA solution (pH 9) or Citric-Citrate solution (pH 6) in autoclave at 120ºC for 20min
- cool down to RT
- permeabilization with Triton 0.2% for 10min
- blocking step with BSA 1% 1h
- primary ab ON 4ºC
- secondary ab 45min
- Hoechst 3342 15min
- mount with prolong
*samples without HIER are just kept in water at RT and Hoechst counterstaining works perfectly
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