[Histonet] Doing double immunofluorescence staining on bacteria, a dilemma, long message

From:Gayle Callis

I have a frustrated researcher whose post doc is trying to stain for a 
protein (antigen) inside the bacteria and a surface antigen. e.g. double 
immunofluorescence.  We know the antibodies work and will stain both, but 
in different circumstances.

The dilemma is

1.  Internalized antigen stains after acetone and acetone/alcohol fixed 
bacteria but surface antigen does not - Tween 20 was added to buffer to aid 

2.  On unfixed bacteria, internalized antigen does not stain, surface 
antigen stains - no detergents are used in buffers.  Obviously the 
acetone/alcohol fixation aided in permeabilization but must have damage the 
surface antigen.  Some antigens do NOT like solvents.

We suspect the surface antigen is compromised by either fixation, detergent 
or both.   The bacterial cell is a lipopolysaccharide in nature, and not 
sure saponin will be much help here either since it is for the cholesterol 
component of cell walls/membranes.

In the past, and before Histonet archives were set up (thank heavens for 
keeping hard copies!) I had this information sent by Peter van de Plas, 
from Aurion in Sept 1998.  He sent this excellent reply to question about 
diffferences between Triton and Tween detergents.

"Both Tween and Triton are non-ionic detergents.  Triton is a low molecular 
weight detergent with high capacity to solubilize lipids/lipid like 
structures. Even after aldehyde fixation you will lose most of your 
membranes and membrane bound proteins.  I would therefore not use it in an 
immunoincubation procedure (you may wash out your antigen.  Tween is a much 
milder detergent.  It is normally used above the critical micell 
concentration  of 0.07%.  the Tween micells will encapsulate antibodies and 
other proteins used in the incubation procedure thus preventing them to 
bind specifically to hydrophobic (sticky) sites in your specimen.  It 
reduces the background by suppressing ctivity of e. g. BSA, normal serums, 
etc.  Like a soap, it will wash all loosely bound protein and it may wash 
out part of your antigen."
Peter van de Plas
Aurion (a company who sells reagents for immunogold staining)


I guess one alternative is to have the internalized protein expressing 
eGFP, and do unfixed bacteria IFA staining for the surface antigen, a nice 
way to eliminate permeabilization and fixation.

Are there any other ways to permeabilize bacterial cell walls?   I don't 
think the researcher is to enamored with trying paraformaldehyde fixation 
if it involves retrievals, etc on these bacteria.  I am not doing the 
staining (my beloved CD markers in frozen sections are easy by comparison!).

Any suggestions will be welcomed.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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