Biology Reference
In-Depth Information
for 2 min, and the supernatants were transferred to clean tubes. Equal amounts of protein
(50 mg) were separated by electrophoresis on a 12% SDS-polyacrylamide gel and
transferred to a nitrocellulose membrane (Millipore, Bedford, MA) using a semidry
electrophoretic transfer system (Bio-Rad). Membranes were incubated in 5% nonfat
dry milk in Tris base/sodium chloride (TBS) buffer with 0.2% Tween 20 (TBST) at
4°C overnight. The membranes were incubated with primary antibodies for 1 h at room
temperature. Blots were washed with TBST and incubated with horseradish peroxi-
dase-linked secondary antibody (Sigma) for another hour, and then proteins of interest
were detected using a chemiluminescence detection kit (Pierce, Inc.).
28.2.7
Immunohistochemistry
Cells were cultured directly on sterile cover slips (Sigma) that were placed into a
6-well tissue culture plate for 2 days. The media was removed from cells, which were
rinsed once with 1× PBS (NaCl 137 mM, KCl 2.7 mM, Na
2
HPO
4
10 mM, KH
2
PO
4
2.0 mM, pH 7.4) at room temperature. The cells were fixed for 10 min at room tem-
perature in 3.7% buffered formaldehyde and washed once in 1x PBS. Fixed cells
were dehydrated by immersing in 70, 95, and 100% ethanol for 5 min each followed
by air drying for 10 min each and stored at 4°C before use. For immunostaining,
samples were rehydrated in decreasing ethanol series (100, 95, and 70%) for 5 min
each. Then samples were immersed in 1x PBS for 5 min at room temperature.
The slides were immersed in quenching solution (3% H
2
O
2
in methanol) for 5 min
and then washed twice in dH
2
O for 10 min. Slides were blocked for 20 min with the
blocking buffer. Primary antibodies (antibody against taurine transporter) were
applied to slides and incubated for 1 h. Slides were washed for 10 min with PBS and
treated with a secondary antibody and incubated for 1 h. After the slides were rinsed
with PBS for 10 min, an ABC reagent was applied for 30 min. Finally, a Metal-
Enhanced DAB Substrate Kit (Pierce) was used to detect immunostaining.
28.2.8
Cell Growth Assay
Control and TauT-deficient 293 cells (1 × 10
4
) were cultured in medium containing G418
(1 mg/ml). The total cell number was quantified every 2 days with a hemocytometer and
an Olympus inverted microscope. Cell viability was assessed by using trypan blue.
28.2.9
Cell Colony Assay
Control and TauT-deficient 293 cells (500 cells per well) were mixed with a tissue
culture medium containing 0.7 agar to obtain a final agar level of 0.35%. Two ml of
this cell suspension was immediately plated in 10 cm plates coated with 0.6% agar
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