I am currently struggling with cell fixing and followed by surface staining. Due to my experimental setup, I had no other choice to fix the cells first and then perform surface staining. I used 2 fixing solution to compare which one works better: 3.75% PFA and BD cytofix solution. Fixation was performed at 4C for 20min. Cells were then washed with cold FACS Buffer. Surface staining with CD80 PE and CD209 BV450 was performed in cold FACS buffer for 20min at 4C. Finally cells were then washed and data was acquired. Fluorescence signals of CD80, CD209 were completely abolished in fixed cells. Unfixed cells showed nice staining of both markers. I am wandering whether fixation was harsh for the surface marker or for the fluorochrome? Would anyone suggest any alternative fixation and stain protocol?
actually the flurochromes you are using are not tandem, so they should not break easily. But even than if you are getting such result than use fixation solution of BD (100 ul in cell pellet) for 10 mins only at 4 degree centigrade. wash with with 1ml or 500 ul PBS (with 1% fetal Calf Serum) at 4 degree centigrade for 8 mins at 1800 rpm. Than re-suspend the cell pellet in desired buffer and immediately perform the flow experiment. all the best
I have not used CD209 in my fixation protocols, but I have had to stain for CD21 and CD23 post fixation. I personally use a solution of 1.6% PFA to fix for 5-10 minutes at 4C. One thing you may want to do is check your PFA to see how old it is, as it can degrade into methanol. Methanol is very harsh and permeabilizes the cells.
I don't know what the problem with your BD cytofix solution could be, I have used it in the past and had no issues. My other advice is to re-titrate your antibodies with fixed cells, as I at times have to double or even quadruple my antibody quantities to get the same fluorescent signal as unfixed cells.
PFA fixation can destroy some epitopes. I would suggest dropping the PFA concentration to 1% or you try fixing in ice-cold methanol (this will permeabilise your cells as well but is less likely to destroy your antibody epitopes)
There is a JOVE site where you can find the video of how to do the surface labeling. The url for that is: www.jove.com/video/51139. You can have the trial version for a month for watching the video.
I found another paper published in Jl of Neuroscience...21(4):1228-1237. 2001
Good luck. I hope it helps.
The problem with comparing immunostaining of unfixed and fixed cells stained with the same antibody is that in many cases the epitopes have been distorted by fixation. There are numerous sources for CD209 for material fixed in paraformaldehyde, so you have to check your specification sheet if your antibody was listed by the company you had bought it from as working on formalin fixed material. If so - check if antigen retrieval was recommended. I have looked ad some antibodies which mention heat induced epitope retrieval (HIER), e.g.you could heck this linklink http://www.merckmillipore.com/GB/en/product/Anti-CD209%2C-clone-EPR5588%2C-Rabbit-Monoclonal-Antibody,MM_NF-MABF749.
Suggested by Lindsey very short fixation in low concentration of paraformaldehyde may work - but you might be disclosing only some of the marker present.
You have mentioned that you have to use fixed cells. Alcohol fixation, as Tara mentioned, is an alternative choice, as you won't need HIER and your antibodies which worked on unfixed cells should (theoretically) work well.
I have concentrated on CD209, the same principles of fixation apply to other markers
Good luck with your work.
Please look into microwave staining methods. See Mathilde Boon or the extra volume in Methods.
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