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ectonucleotidases antibodies
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Western Blot
Protein samples were
resuspended in NuPAGE LDS Sample Buffer (Invitrogen) under non-reducing or
reducing conditions as recommended in the technical data sheet. Note that a number of our Abs do not
recognize the reduced antigen so please avoid DTT and mercaptoethanol when non-reduced
conditions are specified. The proteins
were separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and transferred to
an Immobilon-P membrane (Millipore) by electroblotting according to the
manufacturer’s recommendation (Invitrogen) and then blocked with 2.5% non-fat
milk in PBS-T (136.9 mM
NaCl, 2.7 mM
KCl, 0.15% Tween 20, 10.1 mM
Na2HPO4, 1.8
mM KH2PO4, pH 7.4) for 1 h at room
temperature or overnight at 4°C. After incubation with the primary antibody in 2.5% non-fat milk in PBS-T
for 1h30 at room temperature, the membrane was washed 5 times in PBS-T,
incubated with a horseradish peroxidase-conjugated secondary antibody (1/10,000)
in 0.5% non-fat milk in PBS-T for 1 h, washed 5 times in PBS-T and visualized with
Lightning Western Blot Chemiluminescence Reagent Plus (Perkin Elmer).
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Immunocytochemistry
COS cells were washed twice in PBS (136.9
mM
NaCl, 2.7 mM
KCl, 10.1 mM
Na2HPO4, 1.8
mM KH2PO4, pH 7.4) and then fixed in cold acetone and 10% phosphate-buffered
formalin (19:1) for 2 min at 4°C. After rinsing with PBS, cells were incubated
30 min at room temperature in 7% blocking serum diluted in PBS and then
incubated overnight at 4°C
with primary antibodies diluted in PBS.
Cells were washed twice with PBS-Tween 0.1% and incubated with 0.15%
hydrogen peroxide in phosphate buffered saline for 10 min. Cells were incubated with Avidin/Biotin
blocking kit (Vector Laboratories) for 15 min at room temperature followed by 2
washes in PBS-T and then incubated with a biotin-labelled secondary antibody
diluted 1000 times in PBS for 1 h at room temperature, and followed by 2
washes. The complex avidin/biotinylated horseradish peroxidase (Vector
laboratories) was added for 30 min at room temperature to optimize the
reaction. After washing twice with
PBS-T, peroxidase activity was revealed with DAB (Sigma), 2 to 3 min, as a
substrate. After washing with distilled
water, cells were counterstained with aqueous hematoxylin (Biomeda) as
recommended and mounted in Mowiol.
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Immunohistochemistry
Frozen
sections: Tissues were snap frozen in
isopentane pre-cooled on dry ice/Ethanol, embedded in Tissues-Tek Optimal
Cutting Temperature (O.C.T) freezing medium (Sakura Finetek), and stored at -80ºC until used.
Cryosections (6-8 µM) were mounted on Superfrost Plus slide, fixed in cold acetone and 10%
phosphate-buffered formalin (19:1) for 2 min at 4°C.
Paraffin sections: Embedded paraffin sections were
deparaffinized in toluene and rehydrated in a graded ethanol series (100, 90,
70%) and in distilled water. Slides
were then processed with the antigen retrieval method mentioned in the
corresponding data sheet.
In both tissue embedded procedures, sections
were incubated 30 min at room temperature in 5% bovine serum albumin (BSA)
diluted in PBS and then incubated overnight at 4°C with primary antibody
diluted in 1% BSA. Sections were washed
twice with PBS-Tween 0.1% and incubated with 0.3% hydrogen peroxide in
phosphate buffered saline for 10 min.
Sections were incubated with Avidin/Biotin blocking kit (Vector
Laboratories) for 15 min at room temperature followed by 2 washes in PBS-T and
then incubated with a biotin-labelled secondary antibody diluted 1000 times in
PBS for 1 h at room temperature and followed by 2 more washes. The complex avidin/biotinylated horseradish
peroxidase (Vector laboratories) was added for 30 min at room temperature to
optimize the reaction. After washing
twice with PBS-T, peroxidase activity was revealed with the substrate DAB
(Sigma) for 2 to 3 min. After washing
with distilled water, tissues were counterstained with aqueous hematoxylin
(Biomeda) as recommended and mounted in Mowiol.
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Flow Cytometry
COS cells (0.2-1 million) were resuspended in 100 uL of PFA buffer (1%
fetal bovine serum, 0.1% azide in PBS) and incubated with the primary antibody
for 30 min at 4°C at the recommended concentration specified in the data sheet. Cells were washed
twice by centrifugation at 4000 RPM for 4 min at 4°C with the PFA buffer. Cells
were resuspended in 100 uL of PFA buffer and incubated for 30 min at 4°C
in the dark with a FITC-conjugated secondary antibody diluted 100
times. Cell were washed twice as described previously and analysed with FACS
apparatus.
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Storage
Our polyclonal serums
contain 10% glycerol to preserve IgG functions. To avoid excessive freeze-thaw cycles that affect the antibody
binding capacity, samples can be kept at 4°C for generally up to one
year. Alternatively, the serum can be diluted 10 times for a final
concentration of 145 mM NaCl, 1% BSA, 10 mM Tris, pH 7.4, and 50% (v/v) glycerol.
Samples with 50% glycerol can be kept at -20°C without freezing. Note that 50% glycerol
solution freezes at about -30°C. For long-term storage, freeze at -80°C. |
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