Biology Reference
In-Depth Information
2.
The osmium is washed off well with buffer and the tissue dehydrated by 20 min
incubations in 50%, 70%, 80%, 90%, 95%, two changes of 100%, and two
changes of desiccated 100% alcohol.
3.
The tissue is then placed in propylene oxide for 20 min and then left in 50%
propylene oxide:50% Agar100 medium hardness resin overnight at 4
°
C.
4.
After two changes of Agar100 resin over at least an hour each, the tissue is ori-
ented in specimen vials and the blocks left to harden at 60
°
C for 20 h.
5.
Ultrathin sections can be stained with 2% uranyl acetate (16 min) followed by
3% lead citrate (6 min) with thorough washing in water between and after staining.
3.3.5. Cryoembedding
1.
Pieces of tissue or even the entire gland can be frozen for cryosectioning. The
tissue is frozen in a foil cup of an appropriate size (just bigger than the tissue)
made by moulding aluminium foil over a suitable object such as a marker pen lid
or the cap of a small bottle. This cup is filled with O.C.T mounting medium and
the tissue inside oriented appropriately for sectioning.
2.
The cup is then placed on a metal block precooled in a bath of liquid nitrogen and
left until the O.C.T has become hard and opaque.
3.
Tissue is stored at -20
°
C until required.
4.
Cryosections (7
m) are used for immunostaining using standard fixation and
staining protocols (
Fig 3B
).
µ
3.3.6. Protein/RNA/DNA Analysis
Small pieces of tissue can be snap frozen in aluminium foil parcels in liquid
nitrogen and kept under nitrogen until required. The tissue is then ground down
and protein, RNA, or DNA extracted using standard protocols.
3.3.7. Cell Culture
Mammary epithelial cells can be isolated from transplanted mammary
glands using established protocols
(10)
and their biology studied in tissue culture.
3.3.8. Proliferation Indices
If information on the proliferative index of the mammary gland is required,
then the mouse can be injected ip with a solution of bromodeoxyuridine (BrdU)
2 h before death. This will incorporate into the DNA of any cells in S-phase
during this period. The tissue is then harvested as normal and processed either
for wax embedding and sectioning or cryosectioning and the incorporated BrdU
detected by immunocytochemistry.
3. Notes
1.
Transgenic or knockout mammary epithelium can be serially transplanted once it
has been established that it can form ductal networks within mammary gland.
Search WWH ::
Custom Search