Re: [Microscopy] TEM negative staining issues

[Michael Delannoy]

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Jami,
Is your PTA (phosphotungstic acid, 1% aqueous) adjusted for pH?   After it
solubilizes I use 1N NaOH to get a pH of 7.2, but it only takes a few drops
(4-5).  I have never had issues with fresh PTA, only with old batches would
I see blotches of staining material.  I also filter it (0.22 um Millipore)
as I stain.  It could also be something in your buffer, try running that
alone.  Also try 3 quick D-H2O rinses before the stain.  Remember to do a
complete blot (and get the stain between your forceps with filter paper
triangles).  Check this they talk about staining blobs:

http://science.utep.edu/chemistry/cryo-em/files/PDFs/Negative_Staining.pdf


Sincerely,
Michael  Delannoy

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Subject: [Microscopy] TEM negative staining issues




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I am a relatively inexperienced TEM tech and am having problems with my PTA.
Each time I make fresh PTA, I end up with an image full of stain blobs.
Obviously this is useless. I have tried purchasing fresh PTA, but that
didn't make any difference. I have also used BSA, which helps some, but not
a lot. Help! Any ideas are welcome.

Jami

Jami Carroll
Lab Tech II - Virology
Breathitt Veterinary Center
Hopkinsville, KY
270-886-3959
[hidden email]
 
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