Formaldehyde fixation and EDTA-G decalcification are quite popular methods , however, I got a bad experience with this protocol. Anyone has a successful routine method to fix and decalcified bone samples for immunohistochemistry?
We use a mixture of 4% formaldehyde and 5-10% formic acid for decal of bone-marrow trephines. We compared IHC with EDTA decal and found, that acid decal lead to better IHC. An important point is the sufficient fixation before decal, at least over night.
Another point is, that nucleic acids may be degraded and following analysis may be hampered.
We use Osteosoft from Merck for mouse and rat bones. We tried a lot of options before but this commercial reagent works the best for us
Acid decalcification or chelating agents - the choice depends on the type of antigen, that you will investigate. In my studies I prefer EDTA. The decalcification is longer, but the result is better, compared to fast acid decalcification. I studied VEGF and BMP-2/4 expression. I used rabbits, so my protocol will not suit you. But anyway you will need either X-ray, or chemical test to establish final decalcification.
As Nickolay says, the final test is X-ray or calcium in the bone.
According to my experience 7 days are enough for rat long bones. Simple test is just twisting the bone. If it is flexible it is decalcified. However, fix them well before decalcification.
Thank you Ivan.
In a previous experiment, we have done mechanical tests and it worked. However, X-rays is an excellent option also.
Do you confirm that you just need 7 days to decalcified rat long bones with OSTEOSFT and be successful in immunohistochemistry?
Hi Daniel and Ivan,
I am planning to use Osteosoft for mouse Tibia decalcification because with EDTA I am facing bone marrow shrinkage would able to give me more suggestion like timing or fixation in Formalin is good or should I use PFA?
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