Hi Husne,

I assume that you stain your extracellular markers prior to fixation? In that case it could be a problem of your fluorochromes. APC-Cy7 for instance is known to be quite sensitive to fixation but there are new alternatives on the market that are more stable. You could instead use APC-Cy7 or whichever fluorochrome is giving you problems for the intracellular markers to not expose it to formaldehyde.

If you are staining intra- and extracellular markers at the same time, then try to first stain extracellular, then fixation, then intracellular...

I have heard of people using Saponin only without prior fixation but I haven't tried it myself. I think you should be fine even with fixation.

Good luck,

Jakob

Hi Husne,

I assume that you stain your extracellular markers prior to fixation? In that case it could be a problem of your fluorochromes. APC-Cy7 for instance is known to be quite sensitive to fixation but there are new alternatives on the market that are more stable. You could instead use APC-Cy7 or whichever fluorochrome is giving you problems for the intracellular markers to not expose it to formaldehyde.

If you are staining intra- and extracellular markers at the same time, then try to first stain extracellular, then fixation, then intracellular...

I have heard of people using Saponin only without prior fixation but I haven't tried it myself. I think you should be fine even with fixation.

Good luck,

Jakob

In short yes. Be aware, the stringency of permeabilization is dependent on the stability of your sample and the stringency of fixation. If the sample is unstable, but needs a strong perm then you need a strong fixation or the sample will disintegrate. In contrast a stable sample might handle no fixation and a strong perm. I often use unfixed mammalian cell lines with no fixation and a saponin, triton-X or Tween to stain intercellular and extracellular epitopes in parallel. But I don't know what samples you are using so I don't know if your samples will stay intact, I recommend you try. Keep in mind that without fixation your sample might disintegrate or you might lose epitopes.      

You can do the staining for extracellular marker POST fixation/permeabilization/intracellular staining. In that case, the fixation will not affect the fluorochrom (eg. APC-Cy7). But, you have to be careful if the [extracellular] marker is also expressed and stored intracellularly. In that case, post staining to measure expression of [extracellular] marker is no longer accurate. You can check this by comparing the expression derived from extracellular staining ONLY and staining POST fixation/permeabilization.

The saponin, QuilA bark, diluted to 0.3% in PBS, works very well with most antibodies. Its use does not require to prefix the cells prior to staining.

It is true that the fixing by paraformaldehyde can sometimes alter the recognition of certain antibodies, and in fact it is better to avoid this fixation.

Saponin can be used directly by contacting simultaneously cells, antibodies and saponin: no need for prior contact. By cons it will be enough to extend the contact time and especially to add 1% BSA to cells and antibodies, to avoid harmful aggregation and degradation.

Hi Husne, just to add to the great answers above, when you permeabilize cells without fixation all small soluble cytoplasmic proteins will eventually diffuse out of the cell.

The smaller the cytoplasmic protein is, the faster it will be lost without fixation.

Husne, 

   Not all fixation/perm methods are alike and not all stains work well with all methods.  We have had difficulties with certain surface markers (namely CD19) in the past in our intracellular staining protocols.  We found the best solution was to use a different method of permibilization - utilizing different buffers (MeOH vs. EtOH for example).

Good luck,

Sarah