The slides were then immersed in 10 nmol/L citrate buffer (pH 6.0) and heated in a microwave oven for 5 min to retrieve masked antigens, to reduce nonspecific antibody binding; the sections were then incubated with PBS containing a 10%% solution of normal goat serum and 5%% bovine serum albumin for 30 min.
To reduce nonspecific binding, the tissue sections were then incubated with 5% horse serum (Vector Laboratories) in antibody diluent (DAKO Cytomation, Glostrup, Denmark) for 20 min at room temperature.
In order to prevent non-specific binding of antibodies, sections were then pre-incubated with non-immune mouse serum (1 20; Dakopatts, Hamburg, Germany) and diluted in PBS/BSA (1%) for 25 minutes at room temperature.
To reduce nonspecific binding the tissue sections were then incubated with 5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) in antibody dilutent (DAKO Cytomation, Glostrup, Denmark) for 20 min at room temperature.
To reduce non-specific binding, the tissue sections were then incubated with 5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) in antibody dilutent (DAKO Cytomation, Glostrup, Denmark) for 20 min at room temperature.
Subsequently, 5% BSA in PBS (25 mM Tris, 0.8% NaCl, 2.68 mM KCl (pH 7.4)) was added to block non-specific binding, and the sections were then incubated with a mouse monoclonal anti-FBP1 antibody (1 : 100, Santa-Cruz Biotechnology, sc-374342, Dallas, TX, USA) or anti-c-Myc antibody (1 : 200, Cell Signaling, #9402, Danvers, MA, USA) in a moist chamber overnight at 4 °C.
To block non-specific binding of the primary antibody, all sections were then treated with 100 μl of normal swine serum (NSS) diluted 1 : 20 in Tris-buffered saline (TBS) for 15 min.
Sections were then incubated with horseradish peroxidase-binding amino-acid polymer for 30 min (Histofine Simplestain MAX-PO kit, Nichirei, Tokyo, Japan).
Sections were then rinsed in PBS, and antibody binding was detected by staining with diaminobenzidine/hydrogen peroxidase chromogen solution (DAB+, liquid substrate-chromogen solution, Dako Corporation).
Sections were then incubated with horseradish peroxidase (HRP -binding amino-acid polymer for 30 min (HRP -bindingmplestamino-acid kit, Nichrei, Tokyo, Japolymer
