256
7.
"STAINING"
diluted so that 0.2 N NaOH was present. Relatively long (30 minutes)
staining is indicated. Finally, grids can be lightly carbonized (Chapter 6.5)
before examination in the microscope.
A statistical analysis of the distribution of silver grains is often helpful,
indeed often necessary, to reveal all the information available in autoradiographs. This returns us to the problem that direct resolution is no
better than the size of individual silver grains, which are apt to be rather
larger than mitochondria. Thus, there would be no absolute assurance
that a silver grain was activated by a source from within a mitochondrion
even though it lay directly over it. Statistical methods are required
ordinarily even to prove a correlation of silver grains with organelles
such as mitochondria. It would be quite impossible to differentiate
within mitochondria localization to cristae or to matrix. It can therefore
usually be expected that it will be necessary to classify silver grains in
relation to position in a number of relatively low power micrographs
before valid interpretations as to the distribution of radioisotopes can be
established. The autoradiographic method, valuable as it is and will
prove to be, nonetheless has circumscribed limitations!
7.13. Thick Sections for Conventional Microscopy
The electron microscopist is frequently in the position of wishing to
observe his material with conventional microscopy. His purposes may
range from simply needing an identification of a structure as being
present at the surface of the block, to highly refined microscopy, perhaps
utilizing histochemical techniques. He may wish to combine phase microscopy with electron microscopy.
For the simple identification of structures large enough to be characterized with low power objectives, very crude techniques may suffice.
Even freehand sections may be cut from a block face with a sharp razor
blade and mounted in glycerin or immersion oil under a cover slip, and
then be examined with a 16-mm objective with the condenser diaphragm
nearly closed. Ordinarily, enough will be seen to indicate the tissue
pattern, so that, for instance, glandular alveoli might be seen, a small
arteriole would be apparent, or a glomerulus identified.
The next stage of refinement is to cut appropriately thick sections with
the microtome, which are then picked up on glass. For a mount that is to
be used directly for phase or dark field microscopy rather than for stain-
7.
"STAINING"
diluted so that 0.2 N NaOH was present. Relatively long (30 minutes)
staining is indicated. Finally, grids can be lightly carbonized (Chapter 6.5)
before examination in the microscope.
A statistical analysis of the distribution of silver grains is often helpful,
indeed often necessary, to reveal all the information available in autoradiographs. This returns us to the problem that direct resolution is no
better than the size of individual silver grains, which are apt to be rather
larger than mitochondria. Thus, there would be no absolute assurance
that a silver grain was activated by a source from within a mitochondrion
even though it lay directly over it. Statistical methods are required
ordinarily even to prove a correlation of silver grains with organelles
such as mitochondria. It would be quite impossible to differentiate
within mitochondria localization to cristae or to matrix. It can therefore
usually be expected that it will be necessary to classify silver grains in
relation to position in a number of relatively low power micrographs
before valid interpretations as to the distribution of radioisotopes can be
established. The autoradiographic method, valuable as it is and will
prove to be, nonetheless has circumscribed limitations!
7.13. Thick Sections for Conventional Microscopy
The electron microscopist is frequently in the position of wishing to
observe his material with conventional microscopy. His purposes may
range from simply needing an identification of a structure as being
present at the surface of the block, to highly refined microscopy, perhaps
utilizing histochemical techniques. He may wish to combine phase microscopy with electron microscopy.
For the simple identification of structures large enough to be characterized with low power objectives, very crude techniques may suffice.
Even freehand sections may be cut from a block face with a sharp razor
blade and mounted in glycerin or immersion oil under a cover slip, and
then be examined with a 16-mm objective with the condenser diaphragm
nearly closed. Ordinarily, enough will be seen to indicate the tissue
pattern, so that, for instance, glandular alveoli might be seen, a small
arteriole would be apparent, or a glomerulus identified.
The next stage of refinement is to cut appropriately thick sections with
the microtome, which are then picked up on glass. For a mount that is to
be used directly for phase or dark field microscopy rather than for stain-