210
AKIRA MATSUNO ET AL.
Combined immunohistochemistry and preembedding in situ hybridization at an electron microscopic level
Steps 1 to 11 are the same as described in the section Preembedding Method.
12. Polymerization at 60
0
e for 2 days.
13. The ultrathin sections are attached to Nickel grids.
14. In order to retrieve the immunoreactivity of the targeted
protein, the ultrathin sections embedded in Epon resin
are etched with 10% H20 2 for 30 mins or with 4% sodium
periodate for 10 mins, followed by washing with distilled
water.
15. Immunohistochemical staining is carried out at room temperature for 1 hr using the appropriate antibody.
16. The grids are washed with 0.1 M PB, and the immunoreaction is visualized at room temperature for 1 hr with 20 nm
protein A colloidal gold diluted 1:40 in 0.1 M PE.
17. After being washed with 0.1 M PB and distilled water, the
grids are dried at room temperature, and observed under
an electron microscopy.
The immunohistochemical negative control experiments: substitution of normal serum for the antibody.
Combined immunohistochemistry and preembedding in situ hybridization at an electron microscopic level using LR White resin
Steps 1 to 10 are the same as described in the section under Preembedding Method.
11. 2% osmium tetroxide is applied to the sections. After dehydration with graded ethanol (50%, 70%, 80%, 90%, 95%,
100%), tissue sections are embedded in LR White resin using
inverted beam capsule method.
12. Polymerization at 50
0
e for 2 days in a vacuum oven.
13. The ultrathin sections are attached to Nickel grids.
AKIRA MATSUNO ET AL.
Combined immunohistochemistry and preembedding in situ hybridization at an electron microscopic level
Steps 1 to 11 are the same as described in the section Preembedding Method.
12. Polymerization at 60
0
e for 2 days.
13. The ultrathin sections are attached to Nickel grids.
14. In order to retrieve the immunoreactivity of the targeted
protein, the ultrathin sections embedded in Epon resin
are etched with 10% H20 2 for 30 mins or with 4% sodium
periodate for 10 mins, followed by washing with distilled
water.
15. Immunohistochemical staining is carried out at room temperature for 1 hr using the appropriate antibody.
16. The grids are washed with 0.1 M PB, and the immunoreaction is visualized at room temperature for 1 hr with 20 nm
protein A colloidal gold diluted 1:40 in 0.1 M PE.
17. After being washed with 0.1 M PB and distilled water, the
grids are dried at room temperature, and observed under
an electron microscopy.
The immunohistochemical negative control experiments: substitution of normal serum for the antibody.
Combined immunohistochemistry and preembedding in situ hybridization at an electron microscopic level using LR White resin
Steps 1 to 10 are the same as described in the section under Preembedding Method.
11. 2% osmium tetroxide is applied to the sections. After dehydration with graded ethanol (50%, 70%, 80%, 90%, 95%,
100%), tissue sections are embedded in LR White resin using
inverted beam capsule method.
12. Polymerization at 50
0
e for 2 days in a vacuum oven.
13. The ultrathin sections are attached to Nickel grids.