Biology Reference
In-Depth Information
7.
Goat anti-apoB antibody (Chemicon)
8.
4
SDS-PAGE sample buffer: 200 mM Tris-HCl, pH 6.8, 8% SDS, 40%
glycerol (v/v), 40%
b
-mercaptoethanol (v/v), 0.4% Bromophenol Blue
Other materials and equipment
:
9.
Surgical tools for removing the mouse liver
10.
Motor-driven Potter-Elvehjem tissue homogenizer with loose-fitting Teflon
pestle (Wheaton), 15 ml capacity
11.
Table-top refrigerated centrifuge with swinging-bucket rotor
12.
Beckman ultracentrifuge with SW41Ti swinging-bucket rotor
13.
15 ml Falcon conical bottom tubes
14.
Beckman 13.2 ml Ultra-Clear
TM
or thin-wall polyallomer ultracentrifuge tubes
Additional reagents and equipment for
:
15.
SDS-PAGE and immunoblotting, lipid extraction, thin-layer chromatography
(TLC) plates (lipid analysis), and fast protein liquid chromatography (FPLC)
and Superose 6 (gel filtration column for determining particle size).
7.1.3
Use of BODIPY fatty acids to visualize CLD dynamics
Reagents
:
1.
Collagen solution from calf skin (type I): 0.1% solution in 0.1 M acetic acid
2.
PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
, 2 mM KH
2
PO
4
, pH 7.4
3.
Bovine serum albumin (BSA): essentially fatty acid free (Sigma)
4.
Hepatocyte culture media: Dulbecco's modified Eagle's medium (DMEM),
10% FBS (heat inactivated at 56
C for 30 min), 100 U/ml penicillin, and
100
m
g/ml streptomycin
5.
20
oleic acid complexed to BSA (OA/BSA): 7.5 mM OA, 10% BSA,
dissolved in DMEM (see
Section 2.3.2
. for protocol)
6.
Labeling mediumA: DMEM, 1
OA/BSA, 6
m
MBODIPY FL C
12
or BODIPY
558/568 C
12
(Molecular Probes)
7.
Labeling medium B: QTB fatty acid uptake reagent (Molecular Devices)
reconstituted in 10 ml DMEM containing 1
OA/BSA
8.
BODIPY 493/503 dye: 1 mg/ml in DMSO (Molecular Probes)
Other materials and equipment
:
9.
Spinning-disk confocal microscope with 488 and 543 nm laser or similar
10.
Environment chamber for live-cell imaging with temperature control and CO
2
supply
11.
Stage adaptor and culture chamber for live-cell imaging
12.
Volocity software for image capture and processing
