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)

	Stock Concentration	Volume of Stock
10mM DTT	100%	46.26mg
sdH2O		5.7ml
0.3M NaCl	5M	1.8ml
20mM TRIS, pH8.0	1M	600µl
5mM EDTA	250mM	600µl
1x Denhardt's	100x	300µl
10% Dextran Sulfate	50%	6.0ml
50% Formamide	100%	15.0ml
Total Volume		30.0ml

HB8 Hybridization Buffer (for oligos)

	Stock Concentration	Volume of Stock
10mM DTT	100%	46.26mg
sdH20		9.84ml
1x Denhardt's	100x	300µl
5xSSC	20x	7.5ml
100µg/ml ssDNA	10mg/ml	300µl
100µg/ml tRNA	50mg/ml	60µl
10% Dextran Sulfate	50%	6.0ml
20% Formamide	100%	6.0ml
Total Volume		30.0ml

RNAse Buffer

	Stock Concentration	Volume of Stock
500mM NaCl	5M	100ml
10mM TRIS, pH8.0	1M	10ml
dH20		890ml
Total Volume		1000ml

RNAse Stock (10mg/ml)

10mg RNAse A (Sigma)

1.0ml RNAse Buffer

Heat treat as per Maniatis 1st edition p.451

Working RNAse Solution-20mg/ml

300µl RNAse Stock in 150ml RNAse Buffer

2xSSC, bME, EDTA

	Stock Concentration	Volume of Stock
2x SSC	20x	100ml
10mM beta-mercaptoethanol	100%	875µl
1mM EDTA	250mM	4.0ml
dH20		860ml
Total Volume		1000ml

Box Buffer

	Stock Concentration	Volume of Stock
4x SSC	20x	50ml
50% Formamide	100%	125ml
dH20		75ml
Total Volume		250ml

Stringency Buffer

	Stock Concentration	Volume of Stock
0.1xSSC	20xSSC	20ml
10mM	beta-mercaptoethanol	3.5ml
1mM EDTA	250mM	16.0ml
dH20		3960.5ml
Total Volume		4000ml

Dehydration Buffers:

	50%	70%	90%	100%
100% EtOH	100ml	140ml	180ml	200ml
3M NH4Ac	20ml	20ml	20ml	--
dH20	80ml	40ml	--	--
Total Volumes	200ml	200ml	200ml	200ml

Selected Bibliography

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.

Sources of Materials Used In Situ Hybridization

United Dessicants, 6845 Westfield Ave., Pennsauken, NJ. 08110-1582 USA (609-662-6500)

Humi-Cap dessicant capsules (No. 245-2)

Baxter Scientific (now VWR Scientific)

Nalgene utility boxes (No. L1995-4)

Miles stain dishes (No. S7631-6)

Miles stain racks (No. S7636)

O.C.T. (No. M7148-4)

Tissue culture chamber slides (No. T4135-4)

Peel-a-Way tissue molds (No. M7275-3, No. M7275-2)

VWR Scientific

Micro slide boxes black (holds 25 each) (No. 48444-003)

Slide grips for dipping in emulsion (No. 48440-002)

Polysciences, Inc., 400 Valley Rd., Warrington, PA 18976 USA (800- 523-2575)

Gills hematoxylin No. 2 (No. 4570)

Alcoholic Eosin Y 1% (No. 17269)

International Biotechnologies, Inc.(IBI), P.O. Box 9558, 275 Winchester Ave., New Haven, CT, 06535 USA (800-243-2555)

Kodak NTB2 emulsion (No. 1654433)

Whatman

3mm paper (No. 3030M917)

DE81 filter paper (No. 3658M323)

Fisher Scientific

Autoimmerse heater (No. L1995-4)

Superfrost/Plus Microscope Slides (No. 12-550-15)

Promega Corp., 2800 Woods Hollow Rd., Madison, Wi 53711 USA (800-356-9526)

Riboprobe Transcription buffer kits (ie. No. P2490)

RNasin (No. N2511)

Amersham Corp., 2636 South Clearbrook Dr., Arlington heights, IL 60005 USA (800-323-9750)

35S-UTP (for riboprobe transcriptions) (No. SJ1303 )

35S-dATP (low DTT concentration for tailing rxions) (No. SJ1334)

Sigma Chemical, P.O. Box 14508, St. Louis, MO 63178 USA (1-800-325-3010)

RNase A (No. R5125)

Proteinase K (No. P4914)

tRNA (No. R9001)