I need to perform double immuno EM on sections of tissue embedded in LR White. One of the antibodies has to be an anti-RFP. Does anybody have any previous experience with any anti-RFP on immunoEM? If yes, from which company did you get it, and what is your suggested concentration?
Dear Julian, first of all I would strongly recommend NOT to use LR White. We had a labtech that developed a really nasty allergic reaction over years. If you can, use Lowicryl HM20. If you like, you can have a look in the Material&Methods section of the attached publication. If you have questions, feel free to ask.
Eventhough I do not have specific experience with anti-RFP, I would approach this challenge by finding an appropriate anti-RFP antibody, followed by a gold conjugated secondary antibody. Be prepared to run a titration series for at least the anti-RFP antibody, perhaps keeping the gold-conjugated secondary at a constant and conservative dilution recommended in the spec sheet. The fewer parameters you have, the easier the results will be achieved. Gold luck !
Dear Sacha, even if you are working on mouse tissue you can use antibodies raised in mouse. There are different methods for blocking and reducing background when working "mouse-on-mouse".
Hi Sacha,
I have recently used a RFP ab. from Abcam (raised in rabbits) on legume root nodules infected with bacteria expressing DsRed. The nodules were simply fixed in 2.5% glut and then embedded in LRW (oven-curing). I then used 15 nm Protein-A gold. I'm very pleased with the results - see attached.
Incidentally, I also tried a mouse monoclonal, but that didn't work at all.
Cheers
Sorry Sacha, I just realised that my answer was for Julian!
Cheers
E
Thank you all for your answers! Euan james, could you please let me know the reference number of the Ab from Abcam you used? They actually have 3 different rabbit anti-RFP: ab62341, ab28664 and ab34771 (Although this last one is biotinilated). My guess is that it's the ab62341 but just wanted to make sure.
Hi Julian,
Almost certainly that was it. I performed the immunogold labelling (IGL) just 2 weeks ago, so it would have been one from their current stock. My apologies for my vagueness, but I "borrowed" a range of DsRed/RFP abs to try in IGL, and this was the only one that worked. I'll do some digging to find out exactly which one I used, and I'll get back to you.
I used it at 1:10 in my standard diluting blocking buffer, but you could probably use it less concentrated.
Cheers
E
Hi Julian,
Apologies for the delay, but I can confirm that I used ab62341.
Cheers
E
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