IN SITU HYBRIDIZATION ON FROZEN SECTIONS
Protocol by Josiah N. Wilcox, Ph.D.

1. Remove slides from freezer, thaw for 5 min. at 55°C.
2. Fix 10 min. in 4% paraformaldehyde, 4°C.
3. Wash 5 min. in 0.5x SSC, RT.
4. Immerse slides in proteinase K solution , 1-5 µg/ml in RNase Buffer for 10 min., RT. The amount of proteinase K needs to be optimized with each new preparation. Once optimized aliquots can be frozen down and used for some time.
5. Wash for 10 min. in 0.5xSSC, RT.
6. PREHYBRIDIZATION: Dry around sections with Kimwipe, lay slides flat in an air tight box with a piece of filter paper which has been saturated with Box Buffer (4xSSC, 50% formamide) on the bottom. Cover each section with 100µl of rHB2 without probe (can use 50µl if the tissue is small). Incubate at 42°C for 1-3 hours.
7. HYBRIDIZATION MIX: for 35S-labeled riboprobe.

   Assuming that you have used 100µl of prehybridization buffer combine the following: 2.0µl probe per slide (stock solution 300,000 cpm/µl in 1XTE) 1.0 µl tRNA per slide (50 mg/ml stock) Heat 3min, 95°C immediately add 17.0µl ice cold rHB2 per slide, vortex, place on ice. (Adjust volumes if you have used less than 100ul for prehybridization).

8. HYBRIDIZATION: Add 20µl of above hybridization mix to each 100µl of prehybridization solution directly into the bubble covering the section. Incubate overnight at 55°C. (Adjust volume if you have used less than 100µl for prehybridization).
9. Wash 2 times 10 min. each in 2x SSC with betaMercaptoEtOH-EDTA, RT. (discard to radioactive WASTE)
10. Immerse in RNase A solution (20µg/ml in RNase buffer) 30 min, RT. (discard to radioactive WASTE)
11. Wash 2x 10 min each in 2x SSC with betaMercaptoEtOH-EDTA ,RT. (discard to radioactive WASTE)
12. Wash 2 hours in 4 liters of 0.1x SSC with betaMercaptoEtOH-EDTA , 55°C.
13. Wash 2 times 10 min. each in 0.5x SSC without betaMercaptoEtOH or EDTA, RT.
14. Dehydrate 2 min. each in 50%, 70%, and 90% ethanol containing 0.3M NH4Ac.
15. Dry in vacuum desiccator (3-4 hrs.), store with dessicant until autoradiography.
16. Dip in Kodak NTB2 nuclear emulsion diluted 1:1 with water at 42°C, dry for 2 hours in the dark, expose in the dark at 4°C with desiccant for 2-8 weeks.
17. Develop at 15°C:

    a) 3 min. Kodak D19 developer, diluted 1:1 with water
    b) 20 seconds in water stop rinse
    c) 3 min. Kodak Fixer, full strength
    d) Wash 3 times 5 min. each in water
    e) Counterstain with Hematoxylin and Eosin

Buffers and Solutions

rHB2 Hybridization Buffer (for riboprobes)

HB8 Hybridization Buffer (for oligos)

RNAse Buffer

2xSSC, bME, EDTA

Box Buffer

Stringency Buffer

Dehydration Buffers:

Wilcox, J.N., Gee, C.E., and Roberts, J.L. In situ cDNA:mRNA hybridization: Development of a technique to measure mRNA levels in individual cells. In: Methods in Enzymology, Vol 124, Neuroendocrine Peptides (P.M. Conn, ed.), Academic Press, pp510-533, 1986.

Wilcox, J.N., Smith, K.S., Williams, L.T., Schwartz, S., and Gordon, D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J. Clin. Invest. 82, 1134-1143, 1988.

Wilcox, J.N., Smith, K.M., Schwartz, S.M., Gordon, D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl. Acad.Sci. 86, 2839-2843, 1989.

Melton, D.A., Krieg, P.A., Rebagliati, M.R., and Maniatis, T., Zinn, K., Green, M.R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12, 7035-7056, 1984.