does someone have a protocol about immunofluorescence in bone paraffin-embedded sections? Especially, I would like to know use which method for antigen retrieval?
We have got a section of protocols. I found this one which is OK for you to use even thoght it is with frozen section.
Just use the parafin sections that you have got.
Here is the link:http://www.scientistsolutions.com/index.php?a=protocol&c=431&p=84
Found another one for you:http://www.upstate.com/misc/protocols.q.prot.e.ihcprotocol/Immunohistochemistry+with+Fixed+Paraffin+Embedded+Tissue+Sections
Thank you for you nice reply.
However, bone is a very hard specific tissue, which method can be used for antigen retrieval in the staining?
You can use decalcification to make the bone tissue softer. It helps a lot.
1) 4% PFA ON
2) 5% Formic acid in 2% PFA one week, change fixative daily.
This step can be shorter depends on size of sample and age.
3) 4% PFA 3h-ON
4) regular processing to paraffin embedding
It worked very well in our lab.
Using EDTA to decalcefy the bone tissue could some time interfere with the results.
But I thought that if you used parafin you already decalcified the bone. If not it would be very problematic to cut it.
When I worked with bone I used 10% EDTA and then did frozen section. The protocol that I gave you worked for me very good With anti Osteocalcine.
Thank you for your nice information.
The bone tissues have been decalcified and embedded into paraffin, now I have had lots of bone sections, so my question is about antigen retrieval because we should use some methods (microwave oven, steamer and proteolytic enzyme method) to break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. however bone is very special, i do not know which method is best for antigen retrieval.
maybe this paper will help you:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9075637&dopt=Abstract
Thank you for your article.
We used citric buffer in microwave for 10 min
How many times for the whole process?
how long will it last every time?