246
7.
"STAINING"
been to achieve sufficiently discrete deposition to be meaningful with the
high resolution available with electron microscopy. After all, conventional microscopy can demonstrate an alkaline phosphatase layer in
association with the brush border of kidney proximal tubule cells. There
is no point in extending its investigation to electron microscopic levels of
resolution unless localization can be further delineated as either cytoplasmic, membranous, or extracellular.
Pearse (1963) has discussed many of the practical and theoretical
problems faced by the histochemist who would do his work at electron
microscopic levels of resolution. In tabular form he reviewed through
1961 applications of different techniques. Holt and Hicks (1961) also
have incorporated much information of general interest in a review
article which includes a good bibliography. But certainly most important
at the time of this writing is the paper of Sabatini, Bensch, and Barrnett
(1963) who studied with outstanding success the localization of eight
different types of enzymatic systems in relation to their activities after
preservation with nine different aldehydes (Chapter 3.10). As might have
been anticipated, the character of the fixation proved to be all-important.
This paper also includes a long bibliography.
Insofar as intracellular enzymatic localizations have been concerned,
it usually has been found best to incubate small tissue blocks with a
suitable substrate after an aldehyde fixation, and then often it has proven
desirable to fix secondarily with osmium tetroxide (Chapter 3.10).
Sabatini et al. (1963) have found that once tissue has been preserved in
aldehyde its residual activity can be expected to remain unimpaired for
weeks or months if the tissue is kept refrigerated in a buffered solution.
Thus it can be stored conveniently until ready for use.
In general, tissue loses its enzymatic activity as it is infiltrated and
embedded, particularly if epoxy resins are used. However, Leduc, working
with Holt, reports in private conversation that they have been able to
demonstrate enough residual activity after dehydrating and embedding
in hydroxypropyl methacrylate so that reactions can be produced directly
within thick sections suitable for conventional microscopy (Chapter 4.16).
Unfortunately, activity has not proved adequate so far when attempts
have been made to use ultrathin sections.
Bernhard and his colleagues in Paris have emphasized methods which
utilize embedded and sectioned material as substrates for specific enzymes
that then are added. Once again aldehydes have proved to be the best of
7.
"STAINING"
been to achieve sufficiently discrete deposition to be meaningful with the
high resolution available with electron microscopy. After all, conventional microscopy can demonstrate an alkaline phosphatase layer in
association with the brush border of kidney proximal tubule cells. There
is no point in extending its investigation to electron microscopic levels of
resolution unless localization can be further delineated as either cytoplasmic, membranous, or extracellular.
Pearse (1963) has discussed many of the practical and theoretical
problems faced by the histochemist who would do his work at electron
microscopic levels of resolution. In tabular form he reviewed through
1961 applications of different techniques. Holt and Hicks (1961) also
have incorporated much information of general interest in a review
article which includes a good bibliography. But certainly most important
at the time of this writing is the paper of Sabatini, Bensch, and Barrnett
(1963) who studied with outstanding success the localization of eight
different types of enzymatic systems in relation to their activities after
preservation with nine different aldehydes (Chapter 3.10). As might have
been anticipated, the character of the fixation proved to be all-important.
This paper also includes a long bibliography.
Insofar as intracellular enzymatic localizations have been concerned,
it usually has been found best to incubate small tissue blocks with a
suitable substrate after an aldehyde fixation, and then often it has proven
desirable to fix secondarily with osmium tetroxide (Chapter 3.10).
Sabatini et al. (1963) have found that once tissue has been preserved in
aldehyde its residual activity can be expected to remain unimpaired for
weeks or months if the tissue is kept refrigerated in a buffered solution.
Thus it can be stored conveniently until ready for use.
In general, tissue loses its enzymatic activity as it is infiltrated and
embedded, particularly if epoxy resins are used. However, Leduc, working
with Holt, reports in private conversation that they have been able to
demonstrate enough residual activity after dehydrating and embedding
in hydroxypropyl methacrylate so that reactions can be produced directly
within thick sections suitable for conventional microscopy (Chapter 4.16).
Unfortunately, activity has not proved adequate so far when attempts
have been made to use ultrathin sections.
Bernhard and his colleagues in Paris have emphasized methods which
utilize embedded and sectioned material as substrates for specific enzymes
that then are added. Once again aldehydes have proved to be the best of