Analyzing Cell-ECM Interactions in Adult Mammary Gland by Transplantation of Embryonic Mammary Tissue from Knockout Mice - Methods in Molecular Biology: Extracellular Matrix Protocols - page 331

3. Methods
3.1. Isolation of Embryonic Mammary Tissue
3.1.1. Isolation of Embryos

1. Embryos are obtained by caesarian section from mothers killed by cervical dislo-

cation at the appropriate age of gestation ( see Note 3 ).

2. The ventral flank of the female is opened to expose the bicornate uterus. This is

then gently opened using scissors and fine forceps to reveal the embryos.

3. The embryos should be removed from their foetal membranes and killed by cer-

vical dislocation before dissection. Any remaining attached umbilical cord or
placenta should also be removed, with the aid of a dissecting microscope and
fiberoptic illumination, if necessary.

4. To prevent the skin from drying out, the embryos should be kept moist with

squirts of PBS.

5. For transportation, embryos with heads removed can be shipped in L15 medium

on ice for 24-48 h (but see Note 4 ).

6. The embryonic tail should be removed for genotyping and each embryo given a

unique identifying code to aid correlation with genotype.

3.1.2. Sexing
The sex of the embryos is determined by examining the anogenital distance
which in male mice is larger than in females. Males also have a slight bump
between the urogenital ridge and the anus, which is smaller in the female. In
older embryos (>E15) the lack of obvious nipples in males can also be used to
confirm anogenital sexing. Sexing embryos is quite difficult because the dif-
ferences are small and therefore requires practice ( see Note 5 ).

3.1.3. Removing the Mammary Rudiment

1. The relative location of the nipples in the embryo is the same as in the adult

female ( Fig. 1A ). The mother can therefore be used as a reference aid. The five
pairs of glands lie on either side of the midline in two approximately straight
lines. The first pair are high on the neck (#1 glands; see Note 6 ), pairs two and
three close to the forelimbs, pair four on the abdomen, and pair five in the inguinal
region.

2. A dissecting microscope and adjustable fiberoptic illumination is required to view

the nipples. The embryo should be placed in a Petri dish on its back. A small
piece of tissue underneath the embryo may be useful to prevent it slipping during
dissection. The nipples appear as small white circles on the surface of the skin
( Fig. 1B ). It may aid their location to adjust the angle of the light and move the
embryo from side to side.