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Fig. 3 Analysis of phenotype.
(A)
Haematoxylin and eosin stained paraffin section
of virgin mammary gland;
(B)
Cryosection of virgin transplanted gland stained for
laminin-1. Nuclei counterstained with Hoechst.
4.
Finally, the tissue is cleared for examination with 50% methyl salicylate/50%
alcohol overnight and stored in 100% methyl salicylate.
3.3.3. Wax Embedding
1.
Wax-embedded material can either be obtained directly from fresh tissue or
alternatively pieces of interest can be cut from stained whole mounts and subse-
quently embedded in wax.
2.
If sections are required for
in situ
hybridization, care should be taken to ensure
all solutions are RNase free.
3.
Fresh tissue is fixed in 4% paraformaldehyde in PBS for 1 h at 4
C. Free-alde-
hyde groups are blocked by incubation in 0.2
M
glycine for 2 h at 4
°
°
C.
4.
The tissue is dehydrated through 70% alcohol for at least 2 h followed by 30 min
incubations in two changes each of 90% and 100% alcohol. This is replaced by a
50:50 mix of alcohol:chloroform (30 min), followed by two 45-min incubations
in 100% chloroform.
5.
The tissue is then blotted and transferred to wax 1 at 62
°
C for 10-15 min, changed
into wax 2 (62
C, partial vacuum, 30 min), and finally the vacuum is increased to
maximum for the final 30 min or until no further bubbles emerge from the tissue.
The tissue is oriented in molten wax in moulds and left to harden overnight.
°
6.
m and the sections
mounted on glass slides. Standard haematoxylin and eosin staining protocols can
be used to highlight the histological architecture (
Fig. 3A
).
It is subsequently sectioned on a rotary microtome at 5
µ
7.
Pieces of whole-mounted material require washing with two changes of xylene
over 1 h to remove any methyl salicylate before placing in wax 1 as above.
3.3.4. Electron Microscopy
1.
Small pieces of tissue (1 mm
3
) are fixed overnight at 4
C in EM fix, washed four
times in 0.1
M
cacodylate buffer, and postfixed for 1 h at room temperature in 1%
osmium tetroxide in cacodylate buffer.
°
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