Hi there,
I am working in a project where I do patch clamp recordings in fast spiking interneurons in slices from human neocortex. The electrophysiology is pretty exciting and I need some more anatomical information of my cells. During whole cell configuration I fill the cells with biocytin and subsequently fix them in PFA. Biocytin staining works fine as always, but we have trouble staining for parvalbumin. So we tried out some different parvalbumin antibodies and protocols already. The best one so far is mouse derived (from millipore) and, in rat slices, is probably the best antibody I've used. In my human slices, however, the staining is sparse and after recordings I can't really say if cells are PV-positive or not considering that some wash out of the protein happens during whole cell recordings.
I wanted to ask, if anyone here has experience in staining human parvalbumin and could give me advice on good antibodies and protocols to use?
Thanks in advance!!
Hello Florian, the central problem is that Ca-binding proteins are difficult to detect in slice preparations. Why? Because these proteins are very sensitive against any influences like long lasting staying in incubation chambers . My expiernce was when I did PV-immuno with DAB I just saw fibers no cell bodies. So don't warry about the bad results you have recieved. If you like you can try the pv-antibodies from swant. They are pritty good discribed . I am working with them since more then 20 years. Or you can try the ABs from synaptic systems.
Hi Ute Neubacher , thanks for your answer! Indeed, we have very long storage times for the slices, as the human material can be kept for very long periods. So, when we stain PV directly after slicing, it looks a bit better, but still not satisfying. In rats and mice I don't experience these problems so much, I had slices that I stored for several hours and then recorded from a cell for >1 h, and still PV staining looked great. However, in general our human slices are kept for much longer periods than the rodents', so this is clearly a difference in experimental procedures. Do you know why these proteins are so sensitive against long storage?
Concerning antibodies, we tried one from swant which didn't work unfortunately, but we will try the ones from synaptic systems. Thank you!
Hi Florian, there are several PV-antibodies. A rabbit one is from SWANT (PV28) should be used 1:500. Another is the SySy rabbit one #195002 which we use 1:300.
Hi Hilde, thanks for your answer! So, we tried the rabbit antibody from Swant and it was very disappointing in our human slices. May I ask you what kind of material you used it on?
Hi Florian, sorry that I answered soon. I add 2 publicatons which could help to understand the role and function of PV + interneurons in a better way.