Actually I am asked to stain neutrophils, dendritic cells, T cells in mouse spleen. Do I need to do cardiac perfusion for it?
Perfusion is a good idea if you are planning to do cell specific staining. Otherwise you may encounter big problem with the background staining due to blood/serum present in the tissue. Sometime background is so bad that you will not be able to conclude anything for a cell-specific staining. So yes, perfusion will definitely make it better.
Dear Anuj@ thank you for your suggestion. But the problem is that if i wash the blood, i might lose infiltrating cells such as T cells, neutrophils, dendritic cells. I am not going to do FACS analysis but simply IHC staining (DAB method). Would RBC interfere with the signal in DAB staining? I am really confused. Any suggestion would be so appreciated.
I use the mouse spleen as a positive control for IHC and IF on frozen sections. All I do is collect the spleen and fix it in 4% PFA. You may want to check if your antibodies will work with all fixatives. Also if background is an issue you can always alter the concentration of the blocking solution.
Dear Shyam
If you are doing IHC using peroxidase and DAB inactivate the endogenous peroxidase before, if you don't do this you will have background problems. You can perfuse fix with PFA in PBS. You will lose infiltrating cells only if they are not attached to the endothelium and ready to migrate into the tissue.
Good luck
Background is usually due to serum and that's why perfusion help. I agree with Jaime, you will loose the cells that are infiltrating if they are still in circulation once they have infiltrated the spleen tissue then you should be able to pick up and at that point of time these should be in the tissue matrix protected from being removed while being perfused. RBC should not interfere with DAB. Also make sure your protocol has step for quenching endogenous peroxidase.
Also, after fixation with PFA you can decrease background by incubating in 2 mM Glycine solution in PBS. This would quench the free aldehyde groups that are responsible for some of the non specific fluorescence
Good luck!!!!!!
Thank you all for clearing my doubts. I feel so overwhelmed being among such a gorgeous scientific community.
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