Medha-
We used to use a 0.1% sodium borohydride solution for quenching those
fixatives for tubulin immunofluorescence, but I have not tried it with
an objective lens present. It might be worth a try with a non-precious
objective first, or asking the Olympus support about reactions with
coatings, etc. We used it ice cold, and a bit of gas is released, so
some ventilation is recommended, and small volumes, no sparks or flames.
This would not remove the aldehydes probably, but might reduce
toxicity/reactivity.
You might also quench with a high concentration of tris buffer, which
might result in less toxicity as well.
Tim O'Brien
UNC Chapel Hill
Medha Pathak wrote:
> Hello All,
>
> I'm doing an experiment that requires patching on to a cell, imaging
> fluorescence from a Ca-indicator, fixing the cell while patched and then
> repeating the process with the next sample. I'm having some trouble with
> remnants of the fixative (PFA & glutaraldehyde) either in my recording
> chamber (made of plastic & sylgard) and/or the objective lens affecting
> sample health and patching. I'm using an Olympus 100X 1.1 NA long working
> distance dipping lens. Instead of cleaning just with water, I tried wiping
> the plastic dipping portion of the objective lens with 0.2M ammonium
> chloride. For cleaning the recording chamber, I soak the whole thing in
> ammonium chloride for a few minutes and then rinse with water. This seems to
> work better than water alone.
>
> I wonder whether:
> 1. NH4Cl might be bad for the objective lens coatings. Would it be ok to dip
> the lens in NH4Cl solution?
>
> 2. What other ways can I get the PFA & glutaraldehyde off?
>
> Many thanks for your inputs,
> Medha
>
>
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