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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?