186
J. MILAIRE
observations give a good idea of the trophic aspects of organogenesis.
However, it is necessary to state that histochemical data frequently
differ from one species to the other and may therefore give a somewhat
confused idea of the facts. This is not an embarrassing point, because
such variations are mostly quantitative. We are convinced that more
accurate methods will provide uniform results in more and more classes
of vertebrates.
A. Methods
Ribonucleic acid is demonstrated in sections by the pyronin-methyl
green technique of Unna-Brachet, after fixation with Serra fixative
(90% alcohol, neutral formalin, acetic acid; 6: 3:1). The specificity of this
method is tested by the use of the ribonuclease test on control sections.
Polysaccharides are demonstrated in sections by the P.A.S. reaction
(McManus) after fixation with dioxane or with formalin, both saturated
with picric acid. The localization of glycogen is distinguished from that
of mucopolysaccharides by the systematic use of salivary digestion on
control sections.
Alkaline phosphatase is demonstrated in sections either by Gomori's
cobalt technique or by v. Kossa-Barger's silver method. Two fixatives
are used, both at a temperature between 0 and 4° C. : 80% ethyl
alcohol or 10% neutral formalin in Locke's physiological saline.
Acid phosphatase is demonstrated by Gomori's classical lead technique which recently has been modified for use on embryonic material
(Marit and Milaire, 1961). Tissue blocks (limb buds or small embryos)
are fixed for 16 hours in cold 10% neutral formalin in Locke's solution.
After washing for 24 hours in cold Locke's solution, they are incubated
whole for 2 to 4 hours in a suitable medium. After incubation, they are
washed, dehydrated and embedded in wax in the usual way. The lead
phosphate deposited at the sites of enzymatic activity is then detected
in sections with 1% ammonium sulphide solution. By embedding the
specimens after incubation, rather than before, high enzymatic activity
is maintained in the tissues during preparation.
B. Observations
In all forms considered here, the first proliferation of the dorsal
somatopleure occurs in the whole cephalo-caudal length of that layer,
from the cardiac level up to the cloacal region. The resulting mesodermal
mass raises the ectoderm and modifies the general outlines of the embryo.
This lengthened swelling between the somites and the lateral wall of the
embryo was observed a long time ago in many vertebrates and was named
'Wolff's crest' (v. Baer, 1867 in Braus, 1906). In later stages, the growth
J. MILAIRE
observations give a good idea of the trophic aspects of organogenesis.
However, it is necessary to state that histochemical data frequently
differ from one species to the other and may therefore give a somewhat
confused idea of the facts. This is not an embarrassing point, because
such variations are mostly quantitative. We are convinced that more
accurate methods will provide uniform results in more and more classes
of vertebrates.
A. Methods
Ribonucleic acid is demonstrated in sections by the pyronin-methyl
green technique of Unna-Brachet, after fixation with Serra fixative
(90% alcohol, neutral formalin, acetic acid; 6: 3:1). The specificity of this
method is tested by the use of the ribonuclease test on control sections.
Polysaccharides are demonstrated in sections by the P.A.S. reaction
(McManus) after fixation with dioxane or with formalin, both saturated
with picric acid. The localization of glycogen is distinguished from that
of mucopolysaccharides by the systematic use of salivary digestion on
control sections.
Alkaline phosphatase is demonstrated in sections either by Gomori's
cobalt technique or by v. Kossa-Barger's silver method. Two fixatives
are used, both at a temperature between 0 and 4° C. : 80% ethyl
alcohol or 10% neutral formalin in Locke's physiological saline.
Acid phosphatase is demonstrated by Gomori's classical lead technique which recently has been modified for use on embryonic material
(Marit and Milaire, 1961). Tissue blocks (limb buds or small embryos)
are fixed for 16 hours in cold 10% neutral formalin in Locke's solution.
After washing for 24 hours in cold Locke's solution, they are incubated
whole for 2 to 4 hours in a suitable medium. After incubation, they are
washed, dehydrated and embedded in wax in the usual way. The lead
phosphate deposited at the sites of enzymatic activity is then detected
in sections with 1% ammonium sulphide solution. By embedding the
specimens after incubation, rather than before, high enzymatic activity
is maintained in the tissues during preparation.
B. Observations
In all forms considered here, the first proliferation of the dorsal
somatopleure occurs in the whole cephalo-caudal length of that layer,
from the cardiac level up to the cloacal region. The resulting mesodermal
mass raises the ectoderm and modifies the general outlines of the embryo.
This lengthened swelling between the somites and the lateral wall of the
embryo was observed a long time ago in many vertebrates and was named
'Wolff's crest' (v. Baer, 1867 in Braus, 1906). In later stages, the growth