I am trying to image red blood cells that may or may not have a protein attached to them. We are trying to determine if in fact this protein does attach to the cell membrane. Recently we have been introduced to confocal microscopy and it's uses but there are a few questions lingering about this project and the limitations of confocal.

First being: If we add the protein to red blood cells, incubate, then fix, will this fix any protein associated with the membrane to the membrane so that subsequent washes could be performed without knowing the protein stands a significant chance of being washed away in washes.

Second: If the protein stands a large chance of being washed away and therefore renders it impossible to carry out the steps with the red blood cells in solution is there a way to immobilize red blood cells on a coverslip/plate to then image them?

Thanks for any help you can give!

Hi There,
Welcome to ScientistSolutions
I will try to answer your question but to begin with I need to anderstand what kind of protein you are looking for. Is it attached to the cell ? or you will do it?
Guy

Thanks for the reply. We are looking at the terminal components of the complement cascade that form the Membrane attack complex. Trying to look at a subunit of the complement protein C8. The subunit is the only lipocalin in the complement cascade and as of yet has an unidentified binding partner. The Membrane attack complex of which it is associated with is a mecromolecular complex responsible for pore formation in bacterial membranes as a method of immune defense. We would be trying to ID if gamma does in fact bind to red blood cells (a system used to study complement). We would be adding the protein to the red blood cells then adding antibody and looking to see if it is present on the membrane

Hi again,
Now I understand a bit more about your study.
First of all look at this link and I hope it would help you:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1304820

I can not promise you anything but from my expireance (not with red blood cells) after using 3T3 which attached to a slide coated with Poly-Lysine I managed to do similar things.

What I would do is to try different dillutions of your protein and wash it not under harsh conditions.

I also know that red blood cells could adhare to glas coated by fibrinogen, but am not sure if it would be ok with your experiment, in any case here is a link to a good article:
http://www.jstor.org/pss/40841

Hope this helps.
Guy