H&E on thick ground sections in PMMA

From:Gayle Callis

No possible with any great success on thick, ground sections.  Several
reasons, the PMMA is never totally removed and large dye molecules i.e.
hematoxylin do not work. 

It requires a THIN PMMA section, microtomed, with plastic total removal.  A
thicker PMMA section 40 - 100 um thick section is not going to permit
penetration of dyes throughout, or even on the surface.  1% formic acid
etching is for use with undecalcified bone ONLY in order to remove a few
micrometers of calcium from the BONE (this is a surface decalcification),
it has nothing to do with removal of plastic, and is not going to affect
soft tissues adversely. This is obvious when you look at the section
stained with a T blue or Sandersons rapid bone stain.  The latter is an
oxidized methylene blue, and the original protocol calls for NO acid
etching.  The soft tissues and cells ARE blue to blue green, and
undecalcified bone is counterstained overall blanket red by Van Giesons
with little definition on calcification fronts, canaliculi, Haversian
systems, etc because the calcium is still there.  One you acid etch
undecalcified bone ground sections, the blue bone stain penetrates bone
structures, and Haversian systems show up brilliantly if the section is
left  uncounterstained (VG tends to differentiate out Sandersons from acid
etched bone).  

Softening plastic with alcohol, acetone, or xylene is a solvent etching
which permits better penetration of dyes into the tissues, but this works
best with toluidine blue, methylene blue, basic fuchsin, MacNeals
tetrachrome, light green. All these dyes have low molecular weights and
with higher temperatures and a slightly more alkaline pH (Sandersons bone
stain has high pH)  will stain tissues nicely.   I know of no one who has
had success with thick PMMA sections and an H&E stain. 

If you want an H&E stain, you must cut thin sections i.e. 2 - 6 um thick
with plastic TOTALLY removed, usually by warm xylene for several minutes
and changes. Neil Hand does this beautifully.  In theory, the xylene is
etching the plastic out of tissues, etching is synonomous with removal in
this case and analogous to paraffin removal.  If you have ANY residual
plastic in tissues, dyes will not work as the PMMA is extremely
hydrophobic, staining is weak or spotty.  

Keeping a thick section on a slide is best done with superglue which causes
problems (ugly, diffuse fogged look to section) with H&E staining.  Surface
staining of tissues is best done on white plastic slides in order to view
properly with intense unfiltered light that diffuses up and around the
section to illuminate the surface of the stained section. Surface staining
is not designed for transmitted light through a section as we normally view
an H&E stain on a paraffin section mounted on a clear glass slide. A thin
PMMA section mounted on a glass slide after plastic removal, H&E stained
should work well.  


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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