Biomedical Engineering Reference
In-Depth Information
1. Delipidize slides by incubating them in xylene (rack needs to be mettle) for
5 min at RT twice {
Note *1
}.
2. Remove xylene and rehydrate 1 min each in 100 %, 100 %, 95 %, 95 %, 70 %,
and 50 % EtOH and then in dH
2
O {
Note *1
}.
3. Dry slides well under a hood for at least 2-3 h.
4. Then, in the dark room under red-color safelight, scoop out needed amount of
Kodak NTB2 emulsion into 50 mL tube with the same amount of dH
2
O to make
1:1 ratio solution.
5. Incubate the emulsion solution in a 42°C water bath at least 15 min, occasion-
ally gently mixing but not to make air bubbles.
6. Dip glass slides into the diluted emulsion in the 42°C water bath.
7. Dry dipped slides in a closed lighttight container with silica gel overnight in an
oven at 37 °C.
8. Transfer the slides into the black boxes containing silica gel {
Note *2
}.
9. Seal the edges of the boxes with black electrical tape (avoiding generating static
electricity) and wrap the boxes in aluminum foil.
10. Store the boxes at 4 °C {
Note *3
} from several days to weeks {
Note *4
}.
For Developing Silver Dipping Slides:
11. Warm up the slide boxes to room temperature for 1 h.
12. In the darkroom, set fi ve metal trays with two developer [Kodak, cat#1464593],
one tap water, and two fi xers [Kodak, cat#1971746] and a large plastic tray for
fi nal rinsing with tap water.
13. Develop the dipped slides in Kodak D-19 developer at 16 °C for 3 min, twice.
14. Wash the developed slides in tap water at RT for 1 min.
15. Incubate the slides in fi xer at 19 °C for 3 min, twice.
16. Then, lights can be turned on.
17. Wash the slides in running tap water at RT at least 10 min.
18. While washing, with a razor blade scrape the emulsion off the backside of the
slide that does not contain tissue sections {
Note *5
}, and rinse with tap water.
19. Dry slides up slides at RT.
For Staining Tissues with Cresyl Violet:
20. Stain tissue with 0.3 % cresyl violet in tap water solution for 15 min in a 37 °C
water bath {
Note *6
}.
21. Wash excess cresyl violet solution in fresh tap water for ~15 dips.
22. Dehydrate the slides for ~15 dips each in alcohol solutions: 50 %, 70 %, 95 %,
95 %, 100 %, and 100 % EtOH.
23. Incubate the slides in xylene (rack needs to be metal) for 5 min at room tem-
perature twice.
24. Place Permount medium [Fisher, cat#SP15-500] on the slide, cover slip with a
glass cover slip, and dry the covered slide in the hood for overnight.
25. The sections can be examined underneath the microscope within 1 day, but it
takes about 72 h for the Permount to become hard enough to clean the slide
without the cover slip coming off.
