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Hi everybody!
I'm performing an immunofluorescent detection of the extracellular epitope of a receptor on neuronal cell cultures. I want to quantify the surface expression of this receptor. My antibody recognize an extracellular epitope, so I fix the cells with PFA4%, do not permeabilize the cells with triton neither I use tween20 in the washing steps.
Two questions:
1. A lot of protocols suggest to incubate the primary antibody on the living cells and then to fix them with PFA... Why do not fix cells and then perform the incubation? What are the advantages? Is it possible that PFA also make holes in the membrane (this could explain why sometimes I see also nuclear staining!)?
2. Maybe one could incubate primary and secondary antibody in vitro before adding them together on the cells. This might generate a bigger complex that would have less chances to pass the cell membrane. Why not?
Any suggestions? Thank you
Fri Jul 01, 2011 1:08 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hi sampei
Good Qs!
This is just my opinion...I hope that others will contribute their ideas on this interesting and, imho, controversial area.
1. Yes, you can do live immunostaining: this is suggested ( and confirmed) when you do not wish the fixation to conformationally alter the receptor.
Yes, the PFA will/may make holes in the membrane, altho this is debated.
I have seen excellent evidence showing only extracellular membrane positivity when probing with 2 Abs ( one for external protein: positive, and one for internal protein: negative, when fixing with PFA with no detergent).
However, I have also seen excellent internal positivity after PFA fixation with no detergent.
So, you have to test these variables for your laboratory.
It will take you a week of hard work using appropriate positive and negative controls.
It will be worth it.
2. A good idea! Try this...do not be guided by me
I would suggest that it would not work because if you react primary and secondary as you suggest, the conformational changes would hinder/prevent the complex from optimally finding the Ag. ( steric hindrance)
So, compare live primary ab/secondary Ab then fixation with fixation then conventional Ab detection.
Please keep us informed!
Thanks for the interesting post.
Carl
Sun Jul 03, 2011 7:11 pm
sampei
Rank: Member
Joined: Apr 19, 2006
Posts: 12
Post subject:
Thank you Carl!
I'm currently trying different protocols and I hope to find some evidence to set up a "perfect" method for extracellular epitope staining.
Once I'll get the results I'll post you the news,
thanks again,
Cesare
Mon Jul 04, 2011 4:26 pm
sampei
Rank: Member
Joined: Apr 19, 2006
Posts: 12
Post subject:
Bad news...
I've tried a live staining with 20', 30' and 60' of primary antibody and next the fixation with 4% PFA (to avoid PFA permeabilization) but I only obtained a signal that is not what I expected (maybe it would be the signal of endocytic vescicles)... Do I have to try a shorter incubation time with the antibosy?
I tried also the incubation in Neurobasal medium or in HBSS, no differences.
The in vitro reaction of primary and secondary antibody also give the same results....
Any suggestion? Please...
Mon Jul 11, 2011 4:03 pm
NuclearBlast
Rank: Member
Joined: Jan 25, 2012
Posts: 1
Post subject:
sampei wrote:
Bad news...
I've tried a live staining with 20', 30' and 60' of primary antibody and next the fixation with 4% PFA (to avoid PFA permeabilization) but I only obtained a signal that is not what I expected (maybe it would be the signal of endocytic vescicles)... Do I have to try a shorter incubation time with the antibosy?
I tried also the incubation in Neurobasal medium or in HBSS, no differences.
The in vitro reaction of primary and secondary antibody also give the same results....
Any suggestion? Please...
I would suggest to apply the primary antibody on ice to prevent endocytosis of the antibody. Normally it works for me, with 30' incubation. After I fix the cells with 4% PAF.
I would also have a question though... What do you think, if after washing off the primary antibody I place my cells (primary acy culture) back in heated NB medium, is it possible that I can label the protein-antibody complex after it is endocytosed? How long should I (or can I) keep the cells in the "normal" conditions after the first antibody? Wouldn't be the endocytosed protein-antibody complexes degraded?
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