I am currently trying to differentiate between intracellular and
extracellularly-localized bacteria. I am studying obligate intracellular
parasites, thus I cannot use the gentamicin protection assay. I was
trying to use a 1% paraformaldehyde fixation to crosslink the membrane
and label extracellular organisms and then come back with detergent to
introduce holes before staining the intracellular bugs. I have been
using epi-fluorescence to visualize and photograph the process. Recently
I have been told that the fixation itself is enough to knock large enough
holes in the membrane to allow antibody to stain intracellular organisms
as well as extracellular ones before I ever apply detergent to
solubilize. Can anyone with expertise in the area, give me advise about a
better technique or modification to fit my purpose? I would appreciate your
advise very much.
Sincerely, Julia
Please e-mail me directly at lancekas at u.washington.edu
