protocols for NTPDases antibodies

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).

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.

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.

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.

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.