No significant binding was observed when sections were preincubated with the aminophenyl mercuric acetate (APMA -activated form of MMP (APMA -activatedtMMP-9 (not shown).
The frontal sections were preincubated with 0.3% H2O2 for 20 min to inactivate endogenous peroxidase activity, with 5% NGS for 20 min to block non-specific binding sites and then with 0.1% phenylhydrazine to inactivate endogenous peroxidase activity for 20 min. The sections were then incubated with the antisera against DGKβ (1∶2000) in PBS-T for 72 h at 4°C.
The sections were preincubated for 15 min at pH 10.8 and 5 min at the acid pHs.
Sections were preincubated in a blocking solution of 10% bovine serum albumin (BSA), 0.1% sodium azide and 0.02% saponin prepared in Tris-HCl buffered saline (TBS, pH 7.4) for 30 minutes at RT.
Sections were preincubated for 30 min in 20% H2O2 (Sigma-Aldrich, Spain), and then incubated for 2 h in a solution of 3% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) and 0.3% triton X-100 (Sigma-Aldrich, Spain).
After pretreated with 0.3% hydrogen peroxide in PBS for 15 minutes, sections were preincubated for 1 hour in blocking solution (0.1 M phosphate buffer, 0.25% Triton X-100, and 5% normal goat serum).
Sections were preincubated at room temperature in alkaline stock solution (75 mM glycine, 50 mM CaCl2, and 75 mM NaCl, pH 9.4) adjusted to pH 10.3 with NaOH for 15 min and rinsed three times with distilled water.
Subsequently, sections were preincubated with 1% bovine serum albumin.
After washing sections were preincubated for 1 hr at room temperature in blocking solution 5% NGS in PBS with 0.32% Triton X-100 for blocking nonspecific binding sites.
Tissue sections were preincubated for 30 minutes in the assay medium at 22°C to 24°C.
