I have some problems in IF staining on formalin fixed, paraffin embedded mouse brain tumor tissue.
I tried a protocol that we have in the lab but it seems not works well.. I don't have any signal at all. Do you have any suggestion?
1. Deparafinize Xylen 2x5min
2. Rehydrate 96% EtOH 2x5min
3. Rehydrate 70% EtOH 1x5min
4. Washing 1x PBS 2x5min
5. Citrate boiling 0.01M citrate buffer pH 6.0 (I heat the slides in wicrowave for 5min and than leave it cooled on the bench for 30 min)
6. Washing 1xPBS 2x5min
6. Blocking solution 10% Normal goat serum (NGS)+ 0.1% Triton X-100 in 1xPBS 30min
8. Primary antibody O/N +4°C(in 1xPBS) humidified chamber
9. Washing 1xPBS 1x10 min
10. Secondary antibody R/T 2h (in 1xPBS)
11. Washing 1xPBS 2x5min
12. Counterstaining DAPI 1xPBS
13. Washing 1xPBS 2x5 min
14. Mount the slides with mounting medium and store slides after mounting in +4°C
Thanks in advace!!
Hi Luca Marchetti
Your protocol looks fine! the only problem could be in antigen retrieval.
What antibody are you using?
Heat retrieval is mostly preferred for many antigens using low pH or High pH buffers. But, some epitopes required specific methods i.e., the enzymatic digestion using papain, hyaluronidase etc.,
Please check, if your antibody needs specific treatment for epitope retrieval.
Play with different dilutions of the primary antibody. Incubate the primary ab at room temperature ,not in the fridge. Use, if it is possibel more senssitiv detection systems like biotinylated secondary ab and Streptavidin- flu. Sometimes the green channel (488 nm) shows strong autofluorescence so that a low signal couldn't be seen. Use alexa 545 (CY3) or Alexa 647 (CY5), if you have the suitable filtersystems or lasers for your microscopes. And finally check the datasheet whether the antibody is suitable for this kind of application and for this species which you like to investigate. Good luck!
Dear all thanks for your prompt answer,
Ashis Kumar I am trying the same protocol for this 3 different 1°ab anti TNC-C, NRP1, and p32 (1:200) and we know that the protocol works for TNC-C but on different tissue... not the brain. As you suggest, I will check the data sheet of each antibody to see if there is some info about the antigen retrieval procedure.
But what do you think to heat the slides more... like 10 min and also, should be better pre-heat the citrate buffer and the put the slides or doesn't matter ? I didn't pre-heated the buffer i directly put the slides into the microwaves?
Ute Neubacher I used primary ab concetration (1:200) and the secondary (goat anti rabbit Alexa 647) was (1:500) and I checked the slides on laser confocal microscopy... not signal
I think you can increase the duration of antigen retrieval. Here is my protocol:
4 x 5 min 0,01M citrate buffer pH 6 in the MW; cool 10 min insite the MW; 10 min outsite the MW, 10 min in warm citrate buffer (pour half of the hot buffer away and filled the kuvette or copplin jar up with buffer which you have left at room temperature) . Rinse 3 x 5 in PBS or TBS and continue with your immuno.
Hi Luca Marchetti pre heating the buffer is not going to affect your results. but heating in microwave for 10 mins will over cook your sections and is not advisable. Moist heat (steam) using the pressure cooker is the best.
Hello Luca,
I would suggest to add the tissue in the pre-heated citrate buffer and give 5 to 8 seconds in microwave. Alternatively you may try the water bath, I got good results in water bath for some antibodies . Secondly, wash the samples before and after the antigen retrieval. Lastly some antibodies work better in high concentration. Optimize accordingly.
Good luck!
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