Biology Reference
In-Depth Information
2. Materials
2.1. Isolation of Embryonic Mammary Tissue
1.
Eared (blunt tipped) scissors
2.
Thick forceps
3.
Fine forceps (#5)
4.
Watchmaker's springbow scissors
5.
Dissecting microscope (Leica)
6.
Adjustable fibre optic lights (Euromex, Arnhem, Holland)
7.
Phosphate buffered saline (PBS): 10 m
M
Na
2
HPO
4
, 1.76 m
M
KH
2
PO
4
, 137 m
M
NaCl, 1.33 m
M
KCl pH 7.0
8.
L15 medium (Sigma, Madison, WI, #L4386)
9.
Petri dishes
10.
Cryovials (Nalgene)
11.
Freezing mix (3 parts medium, 1 part serum, 1 part DMSO)
12.
Cryo 1
°
C freezing container “Mr Frosty” (Nalgene)
13.
Glass microscope slides
14.
Telly's fix: 70% ethanol, 5% formalin, 5% glacial acetic acid
15.
Acetone
16.
Ethanol
17.
Meyer's hematoxylin stain for whole mounts: 0.25 g haematoxylin, 50 mg sodium
iodate, 12.5 g aluminium potassium sulphate per liter distilled water.
18.
Acidified 50% alcohol (50% ethanol acidified with 25 mL 1
N
HCl per liter)
19.
Methyl salicylate
2.2. Transplantation of Mammary Tissue into Recipient Mice
1.
Gaseous anaesthetic: Anaesthetic box perfused with 0
2
(2 L/min), NO
2
(1 L/min),
2% halothane
2.
Liquid anaesthetic: One part Hypnorm (Janssen, Beerse, Belgium) and one part
Midazolam (Hypnovel; Roche, Basel, Switzerland) to six parts sterile water for
injection (Fresenius Health Care Group, Basingstoke, UK). Use 10
L/g body
weight by intraperitoneal injection. Anaesthesia should last around 40 min, which
is adequate for a transplantation experiment. If necessary, the period of anaesthe-
sia can be extended by additional doses of 0.3
µ
L/g Hypnorm alone every 30-40 min
and additional doses of Midazolam every 4 h.
µ
3.
Needles (27 gage 1/2 in) and 1-mL syringes
4.
Shaver (Wahl)
5.
Cotton wool
6.
Chlorhexidine gluconate solution (Preston Pharmaceuticals, Preston, UK)
7.
Cork board (Fisons)
8.
Elastic bands
9.
Pins
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