I am setting up a protocol for IF on endomembranes in hippocampal neurons. Thus far, it has been difficult to obtain crisp clear image even when using saponin permeabilization, as it seems that some structures get destroyed, especially the smaller ones. I found that in the literature MES buffer was used instead of PBS. Do you have any experience in that? What could you recommend?
Hi,
To answer your question, I need more details: how do you fix your tissue? Are you using cryosections of paraffin? What is the thickness of your sections? Do all the solution contain saponin? Do you use secondary permeabilization agent?
These are all important details to answer you question correctly. I am not sure changing the buffer will help.
Dear Aleksandra,
I recommend you to use no or 10-fold reduced permeabilisation with detergents. Often due to high viscosity of detergents the concentrations are too high and distrusting membranes more than needed. As well as introducing artefacts, non-specific and diffuse labelling due to disruption and membrane damage. In my experience often in cellular membranes there are enough non-specific pores that allow passage of at least some antibodies across, giving a lot more defined labelling and less diffuse background labelling. If you have to use detergents such as saponin or triton x100, dilute them at least 10-fold more than recommended. I recommend that you also check out my recent methods section below with many hints on this subjects.
Best wishes, Refik
Technical Report Research Methods and Technical Considerations: Answers to ov...
It is not tissue; it's a cell culture on a glass slide, fixed in a very mild 4%PFA and 2% sucrose buffer in PBS. Fixation is not an issue, since overexpression alone produces intact structures (when I mount slides without IF and just after transfection with a fluorescent protein), so something happens afterwards. Respective concentrations used are 0,1% saponin in the permeabilization/blocking buffer and 0,01% saponin in the antibody buffers, with washes in this buffer in between I and II antibody. Triton -X was actually worse. (edit: I went to look at the bench and updated concentrations)
If these are cultured neurons, fixation with 4% PFA for 20 minutes followed by PBS rinse, then chilled methanol, for 3 minutes followed by PBS rinse, should work. Methanol should be kept at - 20 deg C until use and PFA should be made fresh, aliquoted and kept at -20 deg C, and freshly thawed immediately before use otherwise it degrades.
I did post-fix and I like what I'm seeing, thanks :) Looks like the epitope is more visible.
- Badges
- Science topic
- Similar topics
- Biological Science
- Neuroscience