Biology Reference
In-Depth Information
Fig. 2 Clearing the mammary gland of epithelium.
(A)
Diagram of the abdominal
#4 mammary gland indicating the approximate line to cut to remove the distal portion
of the gland containing epithelium. Circle indicated at junction of blood vessels is
lymph node;
(B)
Whole mount of mammary gland from 21-d-old mouse stained with
haematoxylin showing the extent of the ductal network and the darkly stained central
lymph node;
(C)
Stained whole mount of cleared fat pad 6 wk after clearing.
6.
The skin is gently retracted on one side using a sterile swab until the lymph node
of the #4 gland is visible as a small oval in the center of the gland (
Fig. 2A
). To
secure the skin out of the way, a small-gage needle may be used to pin it to the
board. The major blood vessel which forks over the lymph node is cauterized and
the fatty tissue distal to the lymph node is removed using the cauterizer, taking
care not to damage the skin.
7.
The removed tissue can be whole mounted for examination to check that all the
epithelium has been cleared (
see
Subheading 3.3.2.
). On the first few attempts, it
is prudent to leave this cleared gland untransplanted and check after some weeks
that it remains epithelium free (
Fig. 2B,C
). This gives confidence for the future
that any epithelium seen after transplantation is likely to be the result of a suc-
cessful transplant rather than a failed clearing. Once this has been established, it
is preferable to transplant tissue at the same time as clearing the fat pad.
8.
Mammary rudiment, either fresh or thawed (
see
Subheading 3.1.5.
) is placed in
a very dilute solution of trypan blue to aid identification of the tissue during
transplantation. A small pocket is made in the recipient fat pad quite proximal to
the abdomen using fine forceps and the transplanted tissue placed inside using
another pair of fine forceps. The top of the pocket is held against the inserting
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