Another LONG answer on use technics with Sequenza Staining Racks

From:Gayle Callis

To clarify my measurement statement about gaps.  Description of my
experiment, all may find this too detailed/uninteresting. 

First of all, the little gaps YOU describe are actually ledges on which
bottom of slide sits when you mount slide onto coverplate. If you did not
put slide on TOP of these little shelves, they would fall off coverplate,
and I don't think fit into the holding slot.  The gap I talk about is the
space between a plus charge glass slide surface and the flat coverplate
surface with slide resting on top of little shelves ie your "gaps".    

I was always mystified why Shandon recommended 5 minutes rinse after
filling wells when I observed less time for well to be drained. So question
was is there a greater capillary gap between the SLIDE and the actual FACE
OF THE COVERPLATE that permits faster flow = less time.  

The little "gaps" or little shelves you describe were not part of my
equation, and they ARE thicker per your description.  

It is the actual thickness of PLUS CHARGE (little white + marks at bottom
of SLIDE that were factored (as spacers) into pushing a glass slide surface
away from flat face of coverplate, NOT the little shelves (you called these
gaps, but the slides sit on these and should not used as spacers) on the
bottom of coverplates. If you are mounting your slides so that the slides
are pushed away from the coverplate surface by these thicker shelves, then
you are not using the coverplates correctly, and you would get poor
staining and probably drying of sections!  

Erie Scientific provided me with + mark thickness -(marks feel slightly
raised, and a calculation was made based on + mark thickness (hopefully I
didn't mess up! as they gave it to me in a fraction of an inch and had to
do conversion of English to metric, good exercise) Hmmmm think I will ask
them for it again, and redo the calculation or use my fancy dial caliper
for a measurement. 

Next I tried a regular slide (no + marks) on one coverplate  and plus (+)
charge slide on another coverplate, filled wells with buffer and found
there was flow = time difference, concluding the gap created by + marks
permitted buffer to flow through faster. 

Result:  I shortened timing for rinse, less time for overall protocol, and
have excellent staining even with as little as 90 ul, 80 ul is pushing the
limit unless you mount section at bottom of slide. 

Whew, TGIF!



At 03:58 PM 8/2/02 +0200, you wrote:
> Dear Gayle,
>"" which forms the gap but rather there is a gap in the coverblades
>themselves (you can see them and feel them). I also did some quantification
>based on your 147 micrometer:
> length of gap: 5,0 cm
> broadness of gap 2,0 cm
> height: 147 micrmeter or 0,0147 cm
> This makes the volume: 5 x 2 x 0,0147 = 0,147 cm3 = 0,147 cc = 147
> This would mean that if you put on 80-100 microliter antibody, part of
>your sections are not stained or weakly stained (due to diffusion).
> Am I correct or did I misunderstand you?
> A nice weekend to you all!
> jan
>  -----------------------------------------------------
> Jan Bauer, Ph.D
> Div. of Neuroimmunology
> Brain Research Institute
> Spitalgasse 4
> A-1090 Vienna
> Austria
> tel:    +43-1-4277 62813
> fax:    +43-1-4277 9628 
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)


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