[Histonet] Immunofluorescence in bone sections!


Hello, all.


I still have no good results about double immunofluorescence in bone sections until now.

I have modified my protocol according some suggestions, but my results are still bad, only cartilage staining is better. Today my professor shows me some embryo sections, she does double immunofluorescence in embryo sections, and many positive expressions in bone and bone marrow appear. But positive expression is very weak! In addition, I have used the protocol of my professor, but results are also not good. I do not know the reasons, maybe it is related with mouse age and decalcification, but I am not sure of the exact reasons.


this is my protocol:



1.       Not deparaffinization, only dry flat at 37degree to 40 degree for a week.  

2.     Xylol 3x ( every time for 5 min)

3.     Gradient ethanol dehydration ( 99%,99%, 96%, 80%,70% and 50% for 3min)

4.     Distilled water 1x ( every time for 5min)

   Incubate with 0.05% trypsin in 37 degree ( for 15 min)
   Wash with PBS
   Blocking with 5% normal serum matched to the host of secondary antibodies ( for 2h)
   Wash with PBS+0.05%Tween 20  3x (every time for 5min)
   Primary antibody for overnight in 4 degree
   Wash with PBS+0.05%Tween 20  3x (every time for 5min)
   Secondary antibody ( for 2h)
   Wash with PBS+0.05%Tween 20  3x (every time for 5min)
   Nuclear staining (DAPI) for 5min


I do not know what should I do.

If possible, Can you show me your protocol?


Thank you!




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