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

Remove slides from freezer, thaw for 5 min. at 55°C. Fix 10 min. in 4% paraformaldehyde, 4°C. Wash 5 min. in 0.5x SSC, RT. 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. Wash for 10 min. in 0.5xSSC, RT. 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. 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).

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). Wash 2 times 10 min. each in 2x SSC with betaMercaptoEtOH-EDTA, RT. (discard to radioactive WASTE) Immerse in RNase A solution (20μg/ml in RNase buffer) 30 min, RT. (discard to radioactive WASTE) Wash 2x 10 min each in 2x SSC with betaMercaptoEtOH-EDTA ,RT. (discard to radioactive WASTE) Wash 2 hours in 4 liters of 0.1x SSC with betaMercaptoEtOH-EDTA , 55°C. Wash 2 times 10 min. each in 0.5x SSC without betaMercaptoEtOH or EDTA, RT. Dehydrate 2 min. each in 50%, 70%, and 90% ethanol containing 0.3M NH4Ac. Dry in vacuum desiccator (3-4 hrs.), store with dessicant until autoradiography. 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. 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

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.