[Histonet] Re: losing prefixed frozen sections from Plus charge
gayle.callis <@t> bresnan.net
Wed Dec 9 16:46:25 CST 2009
We have suddenly started losing tissue sections from our Superfrost Plus
Our students have been cutting fixed, frozen, cryosections (20um) and
thaw-mounting these onto Superfrost Plus slides. We have suddenly
started losing lots of sections from these slides (again!!). The tissue is
small - ie cross sections of frog aorta and longitudinal sections of
nerves, so any lose of adhesion results in total loss of the tissue. The odd
thing is that she is not losing every section on every slide,
but half to 3/4 of the sections are falling off within the first rinse. The
sections are from the same tissue block on the same slide while some
falls and others don't.
We are at a loss as to what we can do to rescue these sections.
Does anyone know if there is any way to coat the slides in some solution
with the tissue on them to help improve the adhesion without losing the
ability to do immunofluorescence?
Any further advice on cutting / drying protocols are welcomed.
This keeps happening and the inconsistency of it has us so frustrated with
this that we are thinking of going back to subbing our own slides.
1. It could be a bad lot of slides, however, fixed frozen sections are
notorious for falling off slides, even Plus charge. You did not say what
fixative you used either?
2. I did not see where you cryoprotect your fixed tissue prior to snap
freezing, and this may help, not only to reduce large ice water crystal
damage, but also makes sectioning less of a crunchy affair.
3. Dry sections after sectioning in front of a fan at RT, and dry longer.
Do NOT store just cut frozens in the cryostat (you didn't say how you
handled the section immediately after picking up on the Plus Charge slides.
Begin drying at RT immediately after sectioning.
4. Do not touch the surface of slides with fingers, sometime oils from skin
transfer to slide surface.
5. It is a waste to money to coat expensive Plus Charge slides and coatings
ruin or negate the Plus charge. If you carefully read the slide box insert,
you will see this. Do not coat Plus charge slides with anything. If you
want to coat slides with a gelatin (protein) subbing solution or Elmers
glue (derived from milk products), use regular, clean microscope slides,,
coat, air dry and store slides. .
6. Not knowing what or how you rinse, if the rinse is harsh, too fast, it
will wash fixed sections off a slide.
7. 20 um is thick, although people do have success with careful and longer
drying, depending on the staining method you want to perform. How you thaw
this thick section onto a slide may be important. A young man taught us a
clever way to do that by mounting section on a cold slide (cryostat chamber
temperature, then thawing it an angle for a more gradual melting to surface.
(don't put your finger under slide below section so it melts all at once
onto the surface). He started at one edge of the section so it thawed from
one side to another. He had FLAT sections and no bubbles under a thick
fixed section. If your get any unevenness, the section may come up.
Picking up a thick section from the blade holder plate may be part of the
problem so try his method just for fun and possible success. Dry section
8. Fixing the tissue before doing frozen sections compromises the
proteins, and once cross linked, tend to not like to stay on Plus charge
slides, a common complaint seen on Histonet. Hence, subbing your own slides
might be the answer if you had success before.
9. Make sure your blade is sharp so you obtain the flattest section
possible. When sectioning, and you did not elaborate - mount first section
on slide, lay slide outside cryostat, then pick up the next section next to
the first section - we generally put first section closest to label, when
picking up, and work down to bottom (away from label) for the rest of the
sections if you are performing the mounting from the blade holder plate.
Good luck, and be sure to check Histonet Archives for more answers.
Gayle M. Callis
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