General Research Topics > Microbiology

IHC, ICC for mycobacteria


Hi, I am trying to show mycobacterium bovis antigenes in parafin block sections and blood smears. Is there anybody working with immunostaining for mycobacterial demonstration? I seek for advices esp. for Antigen retrieval step. I am using primary antibody against mbovis (Biorbyte) and polymer based detection system (Abcam expose kit anti rabbit). Here is  my protocol:
-Deparafinization, alcohols, AR (110C 10min in autoclave)
-Blocking peroksidase for 10 min, 5min PBS wash, prot block. for 10 min
-Primary ab 1:200 room temp. 1hr,PBS for 5min
-Antirabbit ab 10 min, PBS for 5min
-DAB 10min,
-Counter stain for 30seconds with Harris Hematoxylin
During my trials I had a non spesific nuclear staining. Intracytoplasmic staining in macrophages is expected, I also observe this but there is extra strong nuclear staining which makes it difficult to interpret.  I am going to try different AR steps, in some articles there is even no heat induction at all they been using  enzyme alone. I am going to try different combinations like lower and shorter heat induction, enzyme application or both, I will also try saponin for penetration, I have an information that it is used in cytoplasmic antigens. In some books it is told that for bacterial IHC heat induction is not needed at all. I feel like I have been over retrieving my antigens.
I am busy with IHC in these days and soon will be working with ICC of my samples so any recommendation related to these subjects will be valuable for me. Thanks a lot.

 :'(not helpful but true. Many people used to apply Dako's primary antibody till 2006-2007 but somehow after that they stopped to produce this. It took us a long time to find this Biorbyte's M.bovis antibody but we still couldn't optimize the procedure....


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