I am trying to stain LN sections (from mice), for Madcam-1 using Madcam purified ab as primary and anti-Rat Cy3 ab as secondary. I have tried many times, very few times worked Ok but most of the times the signal was very week inside the organ and Ok in the capsule (we expect to see also in the middle of the LN). Even doing the same protocol and staining steps, many times the signal is extremely week, some other the noise is very high... I have lastly tried to increase the concentration of the primary ab, also the secondary and compared to normal stain (doing the incubation overnight) in all of the samples I get huge noise (even I added different antibodies concentrations).
I really do not know what else to tried because it looks very difficult to visualise this marker inside the lymph node and most of the time I get huge noise or extremely week signal.
Any suggestions? thanks