|From:||Abizar Lakdawalla |
What about bubbles being trapped?
> -----Original Message-----
> From: Gayle Callis [mailto:firstname.lastname@example.org]
> Sent: Friday, August 02, 2002 8:45 AM
> To: Jan Bauer; email@example.com
> Subject: Another LONG answer on use technics with Sequenza Staining
> 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
> 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
> surface with slide resting on top of little shelves ie your
> 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
> >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)
> email: firstname.lastname@example.org