I'm doing an immunofluorescence on suspension cells and after NH4Cl quenching and triton permeabilization steps I'm not able to see the pellet after the centrifugation. At the end of the procedure I usually lose most of the cells. Any suggestions? Someone suggested me to add FBS during the permeabilization. What do you think about it?
Why you do NH4Cl quenching? just curious. If it is part of your immunofluorescence protocol, try to skip this step.
Dear Valeria Lucci ,
Is this immunostaining in live cells or are you fixating them? If you are fixating them with a protein cross-linker (such as an aldehyde), it is ok to apply NH4Cl, otherwise, it is ill-advised to do so.
You can lose cells during immunostaining for several reasons. After permeabilization, the cells slip easily, so could you please elucidate which kind of vials you are using? Are you also removing the supernatants by inversion or through pipetting? It would be best to use polystyrene (not polypropylene) tubes and never perform inversions.
I would consider these points.
Hello,
Is it for microscopy?
If yes, I suggest to coat coverslips with Poly-L-lysine, make your cells attach to it and then make immunostaining like for adherent cells. Like that no more centrifuge at each step.
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