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 !

Sorry, I had forgotten the attachment.....

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