Biology Reference
In-Depth Information
forceps as they are withdrawn to keep the transplanted tissue in place. The trans-
planted tissue should be clearly visible in the opaque mammary fat pad as a small
blue lump. To increase the chances of success, several mammary rudiments may
be transplanted into one recipient fat pad remembering that they should all come
from the same donor embryo to clarify subsequent interpretation of results.
9.
The contralateral #4 fat pad is then cleared and transplanted as above if required.
10.
The skin is sutured closed.
11.
L/g body weight) and left
on a heated pad overnight in a box lined with Vetbed rather than sawdust to recover.
The mouse is injected subcutaneously with analgesia (10
µ
12.
Transplanted mice should be permanently marked or housed individually to aid
subsequent identification.
3.3. Analysis of Phenotype
3.3.1. Harvesting Transplanted Mammary Tissue
1.
Transplanted tissue should be left
in situ
for a minimum of 5-6 wk to see signifi-
cant growth. To stimulate maximum proliferation the recipient animal can be
mated once it reaches 6 wk of age, and the mammary tissue harvested during
late pregnancy.
2.
To access the transplanted glands, the mouse is killed by cervical dislocation and
the ventral skin opened and gently retracted using scissors, forceps and a cotton
swab to reveal the abdominal #4 glands. The distal end of the gland can be sepa-
rated gently from the skin using scissors and, held with forceps, lifted to aid
separation of the rest of the gland from the skin.
3.
Once removed, the entire gland can be spread on a glass microscope slide for
whole mount analysis or dissected into smaller pieces for wax histology, electron
microscopy, cryosectioning, and immunostaining or protein/RNA/DNA analysis.
4.
Alternatively, if passaging the tissue for retransplantation is required (
see
Note 1
), small pieces (approximately 1-2 mm
3
) of gland-containing epithe-
lium (
see
Note 10
) should be cut and frozen (
see
Subheading 3.1.4.
) or immedi-
ately retransplanted.
3.3.2. Whole Mount Analysis
1.
This technique is used on whole gland or pieces of gland to reveal the epithelial
architecture (
see
Figs. 1C
and
2B
). The mammary tissue is gently spread on an
uncoated glass microscope slide and allowed to dry for approximately 1 min. It is
then placed in Telly's fix for at least 2 h.
2.
The tissue is defatted in three changes of acetone (1 h each) and rehydrated
through 100%, 95%, and 70% alcohol (at least 1 h each).
3.
The nuclei are stained with Meyer's haematoxylin for approximately 30 min.
The exact time depending on the age of the staining solution. The haematoxylin
is “blued” in running tap water for approximately 20 min. To improve contrast,
the tissue may require destaining with acidified 50% alcohol before dehydration
through 70%, 95% 2X 100% alcohol.
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