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
|
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)