Bubbles trapped with coverplate method

From:Gayle Callis

This is the hardest thing to perfect - in my estimation -  is mounting the
slide WITHOUT bubbles, and it can be done with practice. Make sure the
coverplate has not been touched by human skin oils - one enemy. 

Wet coverplate well, with a big drop at bottom of slide and coverplate,
mount like a coverslip.  I add goat serum/BSA or some protein carrier to
buffer and also Tween - detergents are notorious bubble makers but help
with sheeting action at this step. 

Pipetting means DO not have bubbles in pipette tips, and I never aspirate
the last little bit. Same for dropper bottles, if they are squirting out
bubbles, trouble!  

Wash Buffer bottles: tips are cut to give a slightly larger diameter hole
for a gentler, wider stream. Make sure lid is on tight, buy bottles that
prevent backwash/bubbles, and introduce buffer by squeezing bottle to start
flow outside holder THEN approach well, and add. That bottle tip must be
filled with bubble buffer.  Direct flow of ALL reagents to BACK of well,
prevent splashing that creates bubbles, and if bubbles occur on top of
filled well, take them off with a plastic pasteur pipette. 

Basically, I do everything I can to prevent trapped bubbles, including a
peroxidase block BEFORE mounting coverplate.  

If bubbles are trapped, they show up during initial slide mounting most of
the time, and if they do, CAREFULLY remount slide.  If you get bubbles
later on, you won't be able to see them UNTIL you finish protocol in your
results, no staining occurs on that area, ugly. 

I know that Pharmingen has used coverplates in past for testing IHC on
their monoclonal antibodies and they have done tons of work. 

Feel like the bubble queen of the day

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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