110
TAKASHI SAWAI AND MIWA UZUKI
Autoradiography
1. Prepare NTB-2 diluted 1:1 with 0.6M ammonium acetate
(4.62g/100 ml, pH 7.4, Prewarm at 45°C)
2. Dip the slides in the emulsion
3. Wipe off any excessive solution on the back of slide
4. Place the slides on a cold stainless steel plate laid horizontally on crushed ice for 3 mins
5. Dry at room temperature for 1 hr
6. Place the slides in the dark box with silica
7. Wrap the box with aluminum foil
8. Place box in the refrigerator at 4°C
Development
1. Develop the slides in Kodak D-19 for 3 mins at I7°C with
intermittent agitation (Exposure time, 3H: 12-14 days, 35S:
5-7 days)
2. Rinse for 30 secs in deionized water
3. Put in Kodak fixative for 2 mins at 18°C
4. Wash the slides ( 2 mins x5)
5. Dry in air
6. Put slides on the plate over crushed ice
7. Stain with the Harris' alum hematoxylin solution (diluted
1:1) for 1 min.
8. Wash in the cold water (1 min x3) ( If the water is not cold,
silver grains come off.)
9. Wash in PBS for 3 mins
10. Wash in cold water (2 mins x3)
11. Dry in air
12. Dehydrate in 100% ethanol
13. Clear in xylene (5 mins x2)
14. Mount with Permout (Inappropriate mount quickly loses
silver grains)