Hi guys,
I would like to do preembedding immunogold on cell cultures. I followed different protocols and I tried to localize different proteins, with both Nanogold (1.4nm) and Aurion (0.8nm) gold particles, and gold enhancement. I never had good results, in particular there was too much background (small gold particles bounded to all membranes).
I will appreciate if somebody could provide to me a good protocol.
Thank you very much!
Francesco
Hi Cecile,
Basically I follow the protocol found in:
Mironov A.A., Polishchuk R.S., Beznoussenko G.V. (2008) Combined video fluorescence and 3D electron microscopy. Methods Cell Biol. 88: 83-95.
- Fix cells in 0.05 % glutaraldehyde plus 4 % formaldehyde in 0.2 M HEPES (pH 7.2–7.3) 30 min RT
- Wash the fixed cells for 3 × 5 min with PBS
- Incubate the cells with the blocking solution (Dissolve 0.50 g BSA, 0.10 g saponin, 0.27 g NH4Cl in 100 ml of 0.2 M HEPES pH 7.2–7.3) for 30 min RT.
- Incubate the cells with the primary antibody diluted in blocking solution overnight at 4 °C
- Wash the cells for 6 × 2 min with PBS
- Incubate the cells with the secondary antibody diluted in blocking solution (1:50-1:100) 2h RT.
- Wash the cells for 6 × 2 min with PBS
- Fix the cells with 1 % glutaraldehyde in 0.2 M HEPES (pH 7.2–7.3) for 5 min
- Wash the cells for 3 × 5 min with PBS and then 3 × 5 min with distilled water
- Incubate the cells with the gold enhancement mixture for 6–10 min. The cells will become violet–gray in color if the gold enhancement is successful.
- Wash cells for 6 × 5 min with distilled water
- Embed in EPON
Thank you very much!
Francesco: would you mind to disclose the molarity (contents, composition, recipe,supplier) of the PBS you use? Hope there will be elucidation on the problem you face... Best wishes and good luck, W.M.
Hi Wolfgang,
I use the 10X PBS stock solution from Sigma P5493
Thank you
Hi,
you can tested many things to reduce your background.
1-decrease the concentration of first antibody
2-you can use BSA-c of orion, it's specific for immunolabelling and can help to deducing the background
another basic questions,
do you tested post embedding ? with this technics you can tested different concentration of antibody and tested different protocols
Hi Francesco,
Have you looked at the Nanoprobes website GoldEnhance™ Tips, Tricks
and Protocols. They list numerous protocols for reducing background.
http://www.nanoprobes.com/newsletters/Vol11_Iss05_goldenhance-tips-enhancement-for-nanogold-in-electron-tomography.html#more
Also I have attached a BBI pamphlet with technical information on gold conjugates which has a trouble shooting section.
Good Luck,
Christine.
Hi Cecile,
I did not tested post embedding. I currently use LRWhite and the ultrastructure is not well preserved
Francesco
Dear Francesco,
I always find LRWhite to be a very extracting resin even in well fixed and non permeablised tissue and so would avoid it wherever possible. See Skepper, J.N. (2000) Immunocytochemical strategies for electron microscopy: choice or compromise. J.Micros., 199(1): 1-36. (http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2818.2000.00704.x/abstract) There is no advantage with pre-embedding immunogold labelling in using LRWhite use Epon as your protocol suggests for best ultrastructure.
- Badges
- Science method