252
7.
"STAINING"
radiographs of tissue from 300 mg salamander larvae which had received
intraperitoneally 1-5 pic of tritiated thymidine. Caro (1961) obtained only
a thinly scattered sprinkling of silver grains after a 2 week exposure of
pancreatic tissue obtained from a 250 gm guinea pig given 2 mc of
tritiated leucine intravenously. In computing reasonable dosages for
whole animals it seems as though 5-10 u.c per gram of body weight of a
tritiated compound with high specific activity is appropriate.
There seems to be general agreement among present workers that the
L-4 Nuclear Research emulsion manufactured by the Ilford Company
offers the best compromise of qualities for purposes of electron microscopy. It can be handled much more easily than any other emulsion with
a comparable grain size, density, and sensitivity (Caro and van Tubergen,
1962). Emulsions with finer grain are available, but so far there have been
difficulties in handling them (Granboulan, 1963). Thus the novice should
start with the Ilford L-4 emulsion, and the description which follows
relates to this.
Sections which are to receive a photographic emulsion should be as
flat as possible. With methacrylate sections, therefore, it is desirable that
they be flattened with xylene vapor (Chapter 5.21). They are then picked
up on coated grids.
The Ilford L-4 emulsion is supplied in a gel form having the shape
and consistency of short pieces of spaghetti. This emulsion is relatively
insensitive to light, and can be handled in the darkroom with a yellowgreen AO safelight filter. Naturally it is best to keep exposures to light
to a minimum. Before use emulsions are stored in a refrigerator, and
have a limited shelf life of only about 4 months after manufacture before
background grain builds up to undesirably high levels.
It is desirable that the silver halide emulsion be spread over tissue
sections as monolayers of crystals. Caro and van Tubergen (1962) have
noted that when liquid emulsion is spread over sections any irregularity
in their surface causes puddling of the emulsion, and uneven thickness
and grain distribution results. Thus they advocate forming a suitably
thin and uniform liquid film of emulsion across a wire loop, and not
applying this to the section until after it has gelled. Hay and Revel
(1963) apparently have not found separate film making to be strictly
necessary with their sections, perhaps because they took more care to
flatten them completely before applying the emulsion by a simple dipping and draining process.
7.
"STAINING"
radiographs of tissue from 300 mg salamander larvae which had received
intraperitoneally 1-5 pic of tritiated thymidine. Caro (1961) obtained only
a thinly scattered sprinkling of silver grains after a 2 week exposure of
pancreatic tissue obtained from a 250 gm guinea pig given 2 mc of
tritiated leucine intravenously. In computing reasonable dosages for
whole animals it seems as though 5-10 u.c per gram of body weight of a
tritiated compound with high specific activity is appropriate.
There seems to be general agreement among present workers that the
L-4 Nuclear Research emulsion manufactured by the Ilford Company
offers the best compromise of qualities for purposes of electron microscopy. It can be handled much more easily than any other emulsion with
a comparable grain size, density, and sensitivity (Caro and van Tubergen,
1962). Emulsions with finer grain are available, but so far there have been
difficulties in handling them (Granboulan, 1963). Thus the novice should
start with the Ilford L-4 emulsion, and the description which follows
relates to this.
Sections which are to receive a photographic emulsion should be as
flat as possible. With methacrylate sections, therefore, it is desirable that
they be flattened with xylene vapor (Chapter 5.21). They are then picked
up on coated grids.
The Ilford L-4 emulsion is supplied in a gel form having the shape
and consistency of short pieces of spaghetti. This emulsion is relatively
insensitive to light, and can be handled in the darkroom with a yellowgreen AO safelight filter. Naturally it is best to keep exposures to light
to a minimum. Before use emulsions are stored in a refrigerator, and
have a limited shelf life of only about 4 months after manufacture before
background grain builds up to undesirably high levels.
It is desirable that the silver halide emulsion be spread over tissue
sections as monolayers of crystals. Caro and van Tubergen (1962) have
noted that when liquid emulsion is spread over sections any irregularity
in their surface causes puddling of the emulsion, and uneven thickness
and grain distribution results. Thus they advocate forming a suitably
thin and uniform liquid film of emulsion across a wire loop, and not
applying this to the section until after it has gelled. Hay and Revel
(1963) apparently have not found separate film making to be strictly
necessary with their sections, perhaps because they took more care to
flatten them completely before applying the emulsion by a simple dipping and draining process.