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We routinely apply Nile Red to PFA fixed samples (green algae, plant tissues).  0.1% Triton slightly disrupt LD morphology, so apply nile red without permeabilization would be preferable if LD morphology is critical.  Also, applying glycerol resulted in high background fluorescence in nile red stained sample. 

李冬齡Tung-Ling Li Chen, Ph.D.
Visiting Associat Fellow
Institute of Plant and Microbial Biology
Academia Sinica



-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Knecht, David
Sent: Tuesday, January 14, 2014 1:05 AM
To: [log in to unmask]
Subject: Re: Nile Red

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I don’t think this is true.  I seem to remember putting live cells in Tween 20 and it had no effect on the cells at all.  We always use Triton to permeabilize.  I agree on the fix.  We never go more than 10 minutes.  Dave

On Jan 13, 2014, at 11:53 AM, Renato Mortara <[log in to unmask]<mailto:[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?

David Knecht, Ph.D.
Professor and Head of Core Microscopy Facility Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)