7.13.
THICK SECTIONS FOR CONVENTIONAL MICROSCOPY
261
paper, McGee-Russell spoke of success in staining Epon-embedded sections with hot (60°C) 1% aqueous Nile blue for 1-2 hour periods.
In general it seems easier to stain Vestopal W sections than epoxy
ones. There are several reports in the literature indicating success.
Gautier (1960) succeeded with a variety of stains including alcoholic
chromotrope, light green, fuchsin, Sudan III, and Lugol's solution. He
also found that aqueous solutions of toluidine blue, eosin, and hemalum
could be used. Thoenes (1960) succeeded with alcoholic Giemsa, and
eosin. Schwalbach et al. (1963) used a variety of aqueous stains including
Mayer's acid hemalum, Hansen's iron-trioxyhematein, Heidenhain's ironhematoxylin, Schneider's acetocarmine, picrofuchsin, and a modified
Giemsa stain. In general, the latter authors simply prolonged the staining time beyond what normally would be used. In commenting upon a
symposium paper by Mercer (1963), Franchi emphasized the use of considerable heat in staining Vestopal sections in a matter of minutes with
such dyes as toluidine blue, pyronine-methyl green, Feulgen reagent, and
for the PAS reaction.
There are of course scattered references in the literature to other stains
being used. I would like to point out from personal experience that the
periodic acid-Schiff reaction can be carried out with most embedments.
We have deliberately used the slowest of the several variant methods to
allow maximum times for the diffusion of substances in and out of the
sections. Thus we have used as an oxidizing bath a 2 hour exposure to
1% periodic acid dissolved in 90% alcohol (Mowry). Orcein works well
with Araldite embedments for elastin.
Recently, Estable-Puig et al. (1964) have revived an old "stain" that
would seem to have much potentiality. It is a specific reaction of
p-phenylenediamine with tissue-bound osmium complexes. They used it
effectively with both Epon and methacrylate sections. Their procedure
was to stain with a freshly filtered 1% solution of ^-phenylenediamine
until the tissue became visibly darkened as brownish-black spots (15-60
minutes, depending upon the thickness of the section and its osmium
content). The slides then were rinsed briefly in distilled water and
dehydrated in two changes of absolute alcohol. This clears the sections
of non-specific background stain that is sometimes seen if an old solution
of p-phenylenediamine is used. Slides then were allowed to dry, and only
after this were they mounted under a cover slip with Permount or other
medium. They advise against the use of a clearing agent such as xylol to
THICK SECTIONS FOR CONVENTIONAL MICROSCOPY
261
paper, McGee-Russell spoke of success in staining Epon-embedded sections with hot (60°C) 1% aqueous Nile blue for 1-2 hour periods.
In general it seems easier to stain Vestopal W sections than epoxy
ones. There are several reports in the literature indicating success.
Gautier (1960) succeeded with a variety of stains including alcoholic
chromotrope, light green, fuchsin, Sudan III, and Lugol's solution. He
also found that aqueous solutions of toluidine blue, eosin, and hemalum
could be used. Thoenes (1960) succeeded with alcoholic Giemsa, and
eosin. Schwalbach et al. (1963) used a variety of aqueous stains including
Mayer's acid hemalum, Hansen's iron-trioxyhematein, Heidenhain's ironhematoxylin, Schneider's acetocarmine, picrofuchsin, and a modified
Giemsa stain. In general, the latter authors simply prolonged the staining time beyond what normally would be used. In commenting upon a
symposium paper by Mercer (1963), Franchi emphasized the use of considerable heat in staining Vestopal sections in a matter of minutes with
such dyes as toluidine blue, pyronine-methyl green, Feulgen reagent, and
for the PAS reaction.
There are of course scattered references in the literature to other stains
being used. I would like to point out from personal experience that the
periodic acid-Schiff reaction can be carried out with most embedments.
We have deliberately used the slowest of the several variant methods to
allow maximum times for the diffusion of substances in and out of the
sections. Thus we have used as an oxidizing bath a 2 hour exposure to
1% periodic acid dissolved in 90% alcohol (Mowry). Orcein works well
with Araldite embedments for elastin.
Recently, Estable-Puig et al. (1964) have revived an old "stain" that
would seem to have much potentiality. It is a specific reaction of
p-phenylenediamine with tissue-bound osmium complexes. They used it
effectively with both Epon and methacrylate sections. Their procedure
was to stain with a freshly filtered 1% solution of ^-phenylenediamine
until the tissue became visibly darkened as brownish-black spots (15-60
minutes, depending upon the thickness of the section and its osmium
content). The slides then were rinsed briefly in distilled water and
dehydrated in two changes of absolute alcohol. This clears the sections
of non-specific background stain that is sometimes seen if an old solution
of p-phenylenediamine is used. Slides then were allowed to dry, and only
after this were they mounted under a cover slip with Permount or other
medium. They advise against the use of a clearing agent such as xylol to