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Thanks,

I was thinking I would do the fixation on poly - l - lysine slides first, then I will remove the cells from the lab followed by staining on the slide.

The protocol I have suggests making a stock in Ethanol. Do you agree?

Shane 

> On 13 Jan 2014, at 18:53, Renato Mortara <[log in to unmask]> wrote:
> 
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> Shane,
> 
> If you fix/permeabilize with Tween, probably all the lipids will be
> extracted. Also, 2h in PFA seems far too much. I would try 30 min, and
> label with Nile Red without permeabilizing.
> 
> Best
> 
> Renato Mortara
> 
> Dr. Renato Arruda Mortara
> Parasitology Division
> Escola Paulista de Medicina - UNIFESP
> Rua Botucatu,  862 6th floor
> 04023-062
> São Paulo SP Brazil
> Phone: 55 11 55798306
> www.ecb.epm.br/~ramortara
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> 
> Dear Group,
> 
> I hope someone can help me with this problem.
> 
> I would like to stain M. tuberculosis cells with Nile Red to investigate
> lipid accumulation. This
> is well published, although literature seems to lack important details.
> 
> My issue is that the cells are required to remain in BSL - 3 lab and our
> microscope is outside
> the lab!
> 
> Could I perform the staining protocol, then fix my cells in 4 %
> formaldehyde (in PBS + 0.05
> % tween 80 (breaks clumps)) for 2 hours (allows for sufficient death of
> cells, thus removal
> from the lab) on poly - l - lysine slide. Then apply glycerol and coverslip.
> 
> My concerns about post fixation is that it will quench or remove the dye?
> Or should I first
> fix, then apply Nile Red?