Re: Bubbles trapped with coverplate method
|From:||firstname.lastname@example.org (Kimberly L Merriam)|
When we add reagents to our slides that are in the coverplates, we use an
eppendorf repeat pipettor (100 ul for reagents, 2 ml for washes). Works like
a charm, and keeps bubbles to a minimum!
Gayle Callis wrote:
> 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: email@example.com
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