7.13.
THICK SECTIONS FOR CONVENTIONAL MICROSCOPY
259
useless. Although Luft (1961) indicated that Heidenhain's iron-hematoxylin could be used with either Araldite or Epon, we have never had
much success with it, even when heroic methods were employed to extract
the cured epoxy resins with sodium methoxide as used by Mayor et al.
(1961) (Chapter 7.13). However, Munger (1961) has indicated his belief
that difficulties in attempting to use hematoxylin relate mainly to the
fact that electron microscopists usually are trying to stain tissues fixed
with osmium tetroxide. He argues that hematoxylin staining is thought
to be dependent on the ionic bonding of a hematoxylin lake to phosphate
groups of nucleic acids. The latter apparently are not available after
fixation with osmium tetroxide, but can be made so by pretreatment with
peracetic acid reagent. The latter is made by mixing 95.6 ml glacial acetic
acid with 259 ml of 30% hydrogen peroxide, and 2.2 ml of concentrated
sulfuric acid. This is allowed to stand 1-3 days, and then 40 mg of
disodium phosphate is added as a stabilizer. This should be stored in a
refrigerator. Munger worked with Epon-embedded tissues, and found it
advantageous also to soak the sections for one hour in xylol before
hydrating them. They were pretreated for one hour with the peracetic
acid reagent, and then could be stained with Ehrlich's hematoxylin in
20-30 minutes. Sections could be differentiated while being observed,
either in tap water or acid alcohol. They could be effectively counterstained with 0.2-0.5% aqueous phloxine B. After this the stain was
drained and the sections were allowed to dry completely. Only then were
they rinsed in absolute alcohol until no more color came out, which was
followed by clearing in xylol and mounting in Permount. A similar pretreatment was used to produce a good aldehyde fuchsin stain, periodic
acid-Schiff stains, and a trichrome stain.
The basic dyes which can be handled most easily, and which prove to
be most generally effective with any embedment, are the Nissl stains,
particularly toluidine blue. Bencosme and his associates (1959) first
emphasized the usefulness of toluidine blue, and pointed out that
metachromasia helps emphasize cytoplasmic differentiation. They used
this dye with methacrylate sections which were first exposed to an acetone
dip that no doubt partially dissolved the embedding medium, and expedited the hydration of the section. After staining with the aqueous dye
they often could see enough detail in temporary aqueous preparations to
satisfy their needs. Permanent preparations could be made in a matter
of minutes by dehydration with acetone and conventional mounting.
Subsequently, Trump and his co-workers (1961) used alkaline toluidine
THICK SECTIONS FOR CONVENTIONAL MICROSCOPY
259
useless. Although Luft (1961) indicated that Heidenhain's iron-hematoxylin could be used with either Araldite or Epon, we have never had
much success with it, even when heroic methods were employed to extract
the cured epoxy resins with sodium methoxide as used by Mayor et al.
(1961) (Chapter 7.13). However, Munger (1961) has indicated his belief
that difficulties in attempting to use hematoxylin relate mainly to the
fact that electron microscopists usually are trying to stain tissues fixed
with osmium tetroxide. He argues that hematoxylin staining is thought
to be dependent on the ionic bonding of a hematoxylin lake to phosphate
groups of nucleic acids. The latter apparently are not available after
fixation with osmium tetroxide, but can be made so by pretreatment with
peracetic acid reagent. The latter is made by mixing 95.6 ml glacial acetic
acid with 259 ml of 30% hydrogen peroxide, and 2.2 ml of concentrated
sulfuric acid. This is allowed to stand 1-3 days, and then 40 mg of
disodium phosphate is added as a stabilizer. This should be stored in a
refrigerator. Munger worked with Epon-embedded tissues, and found it
advantageous also to soak the sections for one hour in xylol before
hydrating them. They were pretreated for one hour with the peracetic
acid reagent, and then could be stained with Ehrlich's hematoxylin in
20-30 minutes. Sections could be differentiated while being observed,
either in tap water or acid alcohol. They could be effectively counterstained with 0.2-0.5% aqueous phloxine B. After this the stain was
drained and the sections were allowed to dry completely. Only then were
they rinsed in absolute alcohol until no more color came out, which was
followed by clearing in xylol and mounting in Permount. A similar pretreatment was used to produce a good aldehyde fuchsin stain, periodic
acid-Schiff stains, and a trichrome stain.
The basic dyes which can be handled most easily, and which prove to
be most generally effective with any embedment, are the Nissl stains,
particularly toluidine blue. Bencosme and his associates (1959) first
emphasized the usefulness of toluidine blue, and pointed out that
metachromasia helps emphasize cytoplasmic differentiation. They used
this dye with methacrylate sections which were first exposed to an acetone
dip that no doubt partially dissolved the embedding medium, and expedited the hydration of the section. After staining with the aqueous dye
they often could see enough detail in temporary aqueous preparations to
satisfy their needs. Permanent preparations could be made in a matter
of minutes by dehydration with acetone and conventional mounting.
Subsequently, Trump and his co-workers (1961) used alkaline toluidine