ectonucleotidases antibodies

Western Blot

Protein samples were resuspended in NuPAGE LDS Sample Buffer (Invitrogen) under nonreducing 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 nonreduced 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 and the bands were visualized using horseradish peroxidase-conjugated secondary antibody incubated in 0.5% non-fat milk in PBS-T for 1 h at a dilution 1/10 000, followed by 5 washing steps in PBS-T and 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 10% phosphate-buffered formalin mixed with cold acetone for 2 min at 4°C.  After rinsing with PBS, cells were incubated 30 min at room temperature in 7% of 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 washed. 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 using 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

Tissues were kept frozen in O.C.T medium (Sakura Finetek) and the section were fixed in 10% phosphate-buffered formalin mixed with cold acetone for 2 min at 4°C.  After rinsing with PBS, tissues section were incubated 30 min at room temperature in 5% of blocking serum diluted in PBS and then incubated overnight at 4°C with primary antibody diluted in PBS.  Section were washed twice with PBS-Tween 0.1% and incubated with 0,4% 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 washed 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 washed.  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 using DAB (Sigma), 2 to 3 min, as a substrate.  After washing with distilled water, tissues were counterstained with aqueous hematoxylin (Biomeda) as recommended and mounted in Mowiol.

 

  

Flow Cytometry

COS cells transfected or not (0.2-1 millions) 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, 4min. 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.