Biomedical Engineering Reference
In-Depth Information
lysosome, and the upstream endocytic pathway (late endosomes,
early endosomes). Other areas of investigation would be to monitor
the effi ciency of fusion of autophagosomes with the lysosome as a
possible source for the inhibition of fl ux. Additional assays can also
be employed to validate the conclusions reached from these prelimi-
nary assays. Interested readers are directed to the recommended
“Guidelines” for such investigations [
7
].
2
Materials
Glass coverslips (pre-cleaned) 13 mm round.
Frosted-edge microscope slides (pre-cleaned).
Poly-
D
-lysine: 1 mg/ml in dH
2
O.
PFA: 3 % paraformaldehyde (diluted from 16 % liquid stock from
Agar Scientifi c) in phosphate buffered saline (PBS), 0.9 mM
CaCl
2
, 0.5 mM MgCl
2
.
Methanol.
CMF-PBS: calcium- and magnesium-free PBS.
PBS-gelatin blocking buffer: 2 % fi sh skin gelatin in CMF-PBS.
50 mM NH
4
Cl in CMF-PBS.
0.2 % Triton X-100 (w/v) in CMF-PBS.
Mowiol 4-88 (Calbiochem) in CMF-PBS (
see
Note 1
).
LC3 antibody (there are numerous suppliers, but we recommend
mouse monoclonal 5F10 from Novus and rabbit polyclonal
cat. No. ab48394 from Abcam).
WIPI2 antibody mouse 2A2 cat. No. ab105459 from Abcam.
LAMP1 antibody mouse cat. No. CD107A from BD.
2.1 For Immuno-
fl uorescence
Microscopy
SDS-PAGE gels, 12 % or ideally Bis-Tris 4-12 % gradient gels
(from Invitrogen) run in MES buffer (Invitrogen).
Lysis buffer: 20 mM Tris-HCl pH 7.2-7.5, 150 mM NaCl, 5 mM
EDTA, 1 % Triton X-100. Complete protease inhibitor
(Roche).
SDS-PAGE reagents and equipment.
5× Laemmli sample buffer: 15 % SDS (w/v), 312.5 mM Tris-HCl,
pH6.8, 50 % glycerol (w/v), 16 %
2.2 For Western Blot
Analysis of LC3-II
ʲ
-mercaptoethanol, a sprin-
kle of bromophenol blue.
Western blot reagents and transfer equipment.
PVDF or nitrocellulose membrane.
Methanol.
CMF-PBS.
