I want to stain an intracellular protein in 4% FA fixed cultured cells. I have to permeabilise the cells. Triton is to harsh (The ultra structure is too much damaged for EM). I need a milder detergent to open up the cells prior to the staining. Saponin is recommended to me.
From previous studies I know that all steps upon permeabilisation w/ saponin require lower conc of saponin too. But I wonder if this holds true for formalin fixed cells. I would like to omit it as the saponin has a bad effect on the ultrastructure.
PS, No, I don’t want to use 0.01% GA as that already damages my epitomes too much.
Hello,
PFA fixation already permeabilises cells. Antibodies can pass the plasma membrane; so they can reach easy-access epitopes but if the epitopes are inside other membranes (nucleus, endocytic compartments...), they will be more difficult to be reached. Just try your immunostaining without detergent.
You mentioned EM. Do you do immunogold staining and/or CLEM?
The immunogold staining without detergent may be difficult as normal colloïdal gold particle size may not pass PFA-only permeabilisation. You may try nanogold particles followed by silver enhancement.
Finally, if you go for saponin permeabilisation, don't forget, you need saponin during all the steps (from permeabilisation to antibody incubations and washes).
Hello!!
If saponin does not work, you can try 0,1 or 0,5% Tween-20 in PBS. We use it for immunoflurescent staining for endosomal system. Tween-20 is a mild detergent, I believe even milder than saponin. It just takes 20 minutes at 37°C and the cells are not damaged afterwards. However, as with saponin, you have to use it for primary and secondary antibody incubation (but not for washes).
I hope everything goes well.
Dear labhelpers,
First of all I would like to thank you for your answers. I read more about the permeabilisation and decided to do a little test, as the papers and manuals are somewhat contradictory.
First of all, I’m working with keratinocytes, quite robust cells. Maybe another cell type does not need permeabilisation (like Barois states), keratinocytes do need it. Only for extracellular epitopes one does not need it. The goal is immuno EM, that rules out glutaraldehyde fixation (immuno won’t work anymore) and also permebilisation with freezing steps, acetone and methanol (ultra structural damage). For Barois, CLEM is not the goal here, I plan to do old fashioned GSSP after DAB staining, that is a technique I have used with good results lately.
As a pilot we cultured keratinocytes and did 3 fixation/permeablisation steps prior to immunostaining of a desmosomal protein. In one staining saponin was added during antibody incubations. Here is an overview of the conditions (FA= formalin; sap = saponin; RT = room temperature)
# : 1 2 3
Fixation (RT) : 20’ 2% FA 20’ 2% FA 10’ 2% FA+0.005% sap
Step 2 (RT): 10’ 0.005% sap 10’ 0.005% sap 10’ 2% FA
Staining (37°C) no sap 0.0005% sap no sap
The result (see image) was that you definitely need saponin during the incubation steps with Ab. In condition 2 we see the expected desmosomal staining. This signal is absent in condition 1 and only faint in condition 3. We haven’t tested saponin included in the washing steps. One paper (Bohn, J Histochem Cytochem 26:4 pp293-297, 1978) stated saponin during fixation was the best for staining and ultra structure. That is tested in condition 3. In this article they show results without saponin during incubation steps.
The concentration of saponin I used was 0.005%.... I must admit I made a mistake there, it should have been 0.05% during permeabilisation and 0.005% during subsequent steps. Though now that I see that the staining works with 0.005%/0.0005%, I will test the EM immunostaining with that conditions. In the near future I hope I can show the EM.
Thanks Duco for your feedback.
In our case, it was primary neurone cell cultures. Without permeabilisation, the immunolabelling at synapses was still good and EM ultrastructure was definitively better than after cell permeabilisation. First we tried CLEM with DAB and now we are trying immunogold labelling but no CLEM.
Happy to see you are making progress in your project.
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