I work with primary cells (explant culture) and while performing IF staining, I require several washing steps. As the culture is a tissue, it seems to get dislodged by the time I perform all the steps and image the sample. Please suggest a good method to prevent this from happening (perhaps a way to reduce the number of washing steps).
In immuno-flourscence studies, you have to follow the washing steps in order to remove the trace of primAB and secAb that otherwise give lots of nonspecific background florescence. Hence, washing is very important steps in order to get the specific signal of your interest and should not be avoided in IHC/ICC.
I would suggest you to give your attention towards the attaching your primary cells using cell adhesion molecules/compounds that are widely used for cell culture system worldwide.
For primary cells adhesion/anchorage, you can use tissue culture grade either type I collagen, gelatin, or fibronectin coated flasks. If you do not have coated flasks, then you can take 0.5 ml of the solution of aforesaid compound and spread uniformly over the culture surface of the flask by the help of scrapper and incubate for 3 h at 37°C. Before the cell culture, individual flasks should be washed with fresh culture medium.
Hope this helps
Best
Instead of growing your cells on dishes, grow
in the wells of 8-chamber tissue culture glass slides (CA62405-178, BD Falcon). Some wells you can use negative controls and some wells you can use Positive controls, you can do 4 immunostaining at a time. You might also able to get 4 chamber wells of 2 chamber wells from the same company,
Also follow the instructions as given how to use these chambers
I have used and they work very well
Good luck
You can use the transwell insert and culture your explants on it. Combined with PDL coating or Collagen IV coating you will have a very robust interface that can even be processed for cryo or paraffin sectioning. We use it for explant co-culture with primary culture monolayers.
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