I'm at a loss here, what I saw before implied the coverplates were being
used as coverslips, could be I was misreading, but here goes a long lecture 
 
Some things learned about using coverplates:

1.	 With plus charge slides, the little plus marks on each edge of slide
end causes slide to be pushed farther away from coverplate.  I asked Erie
Scientific what thickness of this "painted on" plus mark was, calculated
the thickness and found the capillary gap was close to 147 um.  This will
cause your reagents to run through the gap between coverplate and slide
FASTER!  Time your buffer application sometime, Shandon says add buffer,
wait 5 minutes then add next reagent.  I found the buffer left coverplated
slide much faster, 3 minutes was optimal, it did not affect staining
protocol, in fact, shortened time overall. By timing for 3 min, we never
get drying.  WE also add water to bottom of tray to insure proper humidity
chamber.  If you worry about the rinse being too short, do it again, but
the tray will fill up if you do it too much!!   

Buffers MUST flow evenly (all reagents) across the section, some hints coming!

2.     Location of section should be approx half way between label and end
of slide, bottom 1/3 is good.  Too many sections on slide will impede the
flow of buffer, this is a problem with capillary gap Microprobe system too.
 If you insist on more than one section, offset them so the buffer CAN flow
evenly, if this fails, go back to one section per slide.  Sections must be
flat, without wrinkles for best flow. If you get no sheeting of buffer,
section could dry, and you can't see the section in that holder.  It would
be nice if Shandon would make the tray bottom a clear plastic! 

3.	  One lab had this problem by circling the section with Pap pen before
applying the coverplate, DO NOT DO THIS, the hydrophobic pap pen mark will
impede reagent flow. I did not perceive this was your problem - just
something to be aware of.  

4.	  Use Tween or another detergent, this helps reagent flow AND give
sheeting action in that tight space between section/coverplate. 

5.	  Make sure coverplate is not touched by your fingers, oil from skin
messes up the surface, preventing good sheeting action. Removed coverplates
are immersed immediately into distilled water, never allow serums, etc to
dry on surface, then wash by using a heavy stream of water, place
coverplates in slanted position to dry, and rinse with distilled water post
wash.  We do not use any soap for washing.  Any scratched surface near
section area means tossed coverplate, the scratching at bottom where slide
first comes in contact is not a big factor

7.	  Be sure coverplate has a goodly amount of buffer on it BEFORE mounting
the slide, bubbles must be totally absent. We like to use a buffer
containing detergent and some normal serum or BSA.  This helps slide
mounting without bubbles.

8.	If the coverplate permits buffer to run TOO FAST, toss the coverplate,
it is probably flawed 

 
AS for mounting media, try Crystal Mount or one of its equivalents, this is
a liquid coverslip to insure good section coverage - you should be able to
visualize its coverage easily, then mount a permanent coverslip after
Crystal Mount is totally dry.  

I have trouble with aqueous mounting medias retracting during drying, I use
more, and do not overly blot away excess.  I have also sealed the edges
with diluted mounting media, not fingernail polish which contains alcohol
and can leach into aqueous media.  Crystal mount would be a better
alternative for this messy procedure.  

Whew, what a blab session

  

 

At 10:54 AM 1/9/02 -0500, you wrote:
>Interesting! I have been having some difficulty with non-aqueous mounting
>media and did not think of the coverplates as a possible problem. (drying
>starting in the center of the mounted section after few weeks in a "fern"
>type pattern)  I thought that the dehydration, xylene or the mounting media
>itself was a problem and have changed and adjusted number of stations in the
>dehydration, changed media and so on.  The problem has improved, but still
>will happen on occasion.  I have noticed that it will happen with human
>brain tissue but NOT the mouse brain ran on the same system as you have
>(Shandon coverplate/non automated system).
>
>Could any of the reps for Shandon give us feedback on any changes in the
>Coverplates in the last few months?
>
>Rose
>
>
>----- Original Message -----
>From: "Mikael Niku" 
>To: "'Histonet'" 
>Sent: Wednesday, January 09, 2002 6:09 AM
>Subject: Mounting problem with Coverplated slides
>
>
>Dear histonetters!
>
>We recently started using Shandon Coverplates for
>immunohistochemistry and in situ hybridization (without
>the staining center or automatic stainer).
>
>Everything seemed to be fine first and we were very happy
>with the system. However, we soon noticed that using
>Coverplates resulted in mounting problems. We are using
>an aqueous mounting medium (traditional glycerin-gelatin
>jelly) as the NBT/BCIP stain of in situ hyb seems to
>fade with xylene-based stuff. This used to work just well,
>but with Coverplated slides the mounting medium seems
>to evaporate or contract somehow, and sections often
>get dehydrated (and thus ruined). This happens very quickly,
>in a matter of hours or days.
>
>I can't understand why this might be. The slides don't
>even go directly from Coverplates to mounting, but they
>are at least washed a few times and counterstained and
>again washed in between. But this really happens exactly
>and only with slides that have been stained using Coverplates.
>
>Any ideas, or better yet, suggestions for solutions?
>
>\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
>   Mikael Niku             URL: www.helsinki.fi/~mniku/
>   University of Helsinki  Dept. Basic Veterinary Sciences
>       - Mitäkö mieltä olen länsimaisesta sivistyksestä?
>         Minusta se olisi erinomainen ajatus!
>                                              - Gandhi
>////////////////////////////////////////////////////////////
>
>
>
>
>
>
>
>
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)