I want to transfect my hippocampal neurons (cultured in Neurobasal medium on Poly-D-Lysin coated wells) with labeled antibodies.I followed this protocol (with some changes):
For those of you who can't see the protocol, here a short summary:
The antibodies are labeled with Atto550 NHS, dialyzed (in PBS) and concentrated (with spin columns). Then the labeled antibodies are mixed with medium and Ab-DeliverIN reagent and the cells are transfected with this mix. After 48 hours, the cells are fixed, washed 4 times 5 minutes with PBS and studied with a fluorescence microscope.
The transfection actually worked on the first try, but there is a lot of background staining, e.g. the whole bottom of the the wells are stained in red (except where the cells are, since they are stuck on the well). Of course with this high background it is nearly impossible to make certain statements of the binding. We are now trying to get rid of this background and we thought that when we add 4% BSA to the cells shortly prior to transfection, the bottom of the well should be blocked and background staining should be minimized.
The question is, will BSA interfere with the transfection mechanism, or will it damage the cells? For the next transfection we will also increase the washing time to 2 hours (changing the PBS every 20 minutes) and maybe we will change the medium 24 hours after transfection, to get rid of not transfected antibodies.
And the general question:
How often can I freeze and thaw the BSA, before it goes bad? We want to filtrate our BSA stock with sterile filters and then freeze them again.
Greetings from Germany!
Edited by DonPromillo90, 18 April 2013 - 08:48 AM.