Antigen retrieval was performed by incubation for 10 min at 121°C in citrate buffer (0.01 M, pH 6.0); sections were washed with phosphate-buffered saline (PBS), and incubated for 30 min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK).
After preincubation, the tissue sections were washed in binding buffer (50 mM Tris-HCl buffer, pH 7.4) and air-dried.
The sections were washed with buffer and counterstained in 1% aqueous uranil acetate and lead citrate for 30 min and 1 min respectively, and examined by a JEOL 1200 EX transmission electron microscope.
After, sections were washed in buffer and immunodetection was performed by treatment with the chromogenic 3 3 diaminobenzidine (DAB) solution.
Sections were washed in buffer and a peroxidase reaction product was localized 3,3'-diaminobenzidine tetrahydrochloride in the presence of H2O2.
Following the detection protocol described by the manufacturer, sections were washed with buffer A at room temperature and incubated with the ligation solution for 1 h at 37 °C.
Sections were washed in Buffer 1 for 6 × 30 min. Detection of probes/anti-DIG antibody was achieved by addition of NBT/BCIP solution (Roche; 20 μl/ml) in 0.1 m Tris (pH 9.5)/0.1 m NaCl (Buffer 2).
Antibody mixture was removed by aspiration, sections were washed sequentially with buffer 1 (0.25% TX-100, 1x PBS) and buffer 2 (1x PBS).
Sections were washed in rinse buffer and incubated for 5 min in blocking buffer (1% bovine serum albumin (BSA) in Tris-buffered saline (TBS)).
The sections were washed with HEPES buffer containing milk powder and gammanorm (5 min, 4 °C), 3× HEPES buffer (5 min, 4 °C), and finally 2× distilled water (10 s, 4 °C) and were dried at rt. Standards of 1 μL tracer solution dried on 0.87 cm2 filter papers were prepared as positive controls.
