1. Harvest tissue and rinse in PBS or saline.
2. Immerse tissue in 4% paraformaldehyde/0.1M sodium phosphate buffer pH7.4 (recipe follows) at 4°C for 1-3hrs. Try to avoid overnight fixation if possible as this causes problems with tissue adherence on slides during the hybridization procedure.
3. Immerse tissue in sterile 15% sucrose/1xPBS (recipe follows) 3 hrs. to overnight at 4°C.
4. Embed tissue in O.C.T. (Baxter No. M7148-4), M1 (Lipshaw) or any other convenient embedding matrix for frozen sectioning in plastic embedding molds (. Tissue should be oriented in the block appropriately for sectioning (cross-section, longitudinal etc.). Note the tissue number on the block directly for reference.
5. Freeze tissue block in liquid nitrogen. Place the bottom third (aproximately) of the block into the liquid nitrogen, allow to freeze until all but the center of the O.C.T. is frozen, and allow freezing to conclude on dry ice.
6. Store tissue blocks at -70°C in a sealed container or wrapped in foil and ship on dry ice.

It is also possible to use fresh frozen tissue for in situ hybridization if the paraformaldehyde/sucrose method is not feasible. Tissues should be rinsed in saline or PBS and frozen in O.C.T. blocks in liquid nitrogen as outlined above. Although not optimal, it is also possible to use snap frozen material tissue without an embedding matrix. The fixation, sucrose, and O.C.T. steps are used primarily to improve the tissue morphology.

It is expected that the fixation times outlined above will not result in complete fixation of large pieces of tissue. However, the additional fixation step at the beginning of the in situ hybridization procedure should ensure adequate fixation of such tissues prior to hybridization.

This protocol has been used successfully on large (up to 1 cubic cm) and small (1 cubic mm) tissue samples.

4% Paraformaldehyde

Mix in a two liter flask:

200ml 0.5M NaPO4, pH 7.4
800ml depcH20
Heat to 70°C with stirring on hot plate in fume hood
Add 40g Paraformaldehyde (EM grade, Polysciences, Cat No. 0380)

Once the solution has cleared (it should take 5 minutes or less), filter with a side-arm flask, Buchner funnel and Whatman No. 2 filter paper.
Immediately pour the solution into a one liter bottle which has been packed in ice. This cools the solution quickly and prevents breakdown of the paraformaldehyde. Store at 4°C for up to two weeks.

15% Sucrose in PBS:

500ml sterile PBS
75g "RNase free" sucrose

Mix above and filter sterilize with a disposable Nalgene filtration unit type S(0.45 micron). Store at 4°C.

USE OF FISHERBRAND SUPERFROST/PLUS MICROSCOPE SLIDES FOR IN SITU HYBRIDIZATION

We use Fisherbrand SuperFrost/Plus positively-charged microscope slides (Cat. No. 12-550-15) for all of our frozen tissue sectioning and have very good tissue retention on slides after an in situ hybridization experiment. SuperFrost/Plus slides require no preparation time prior to cryosectioning and are competitive in terms of labor cost and reagent expenses.

SECTIONING OF FROZEN TISSUES FOR IN SITU HYBRIDIZATION

1. Frozen tissues prepared as described can be wrapped and stored for many years prior to sectioning, without loss of the mRNA signal. The biggest problem with stored tissue blocks is that they tend to dessicate if not properly wrapped and the O.C.T. (Optimal Cutting Temperature compound) can be difficult to cut.
2. Blocks should be removed from the -70°C freezer and allowed to equilabrate with the cryostat chamber temperature. Tissues can be cut at any convenient temperature (-15 to -35°C) as needed. Most tissues cut well at -15°C (brain, kidney, liver, vessels, muscle, etc.) however fatty or more difficult tissues (adipose tissue, skin, lung) require temperatures as low as -35°C or less to obtain good sections. Care should be taken not to touch the face of the slides but handle by the edges only. Frozen sections 5-7µm (thinner is OK but thicker, over 10µm, may present problems for visualizing mRNA in situ) should be cut, thaw-mounted onto room-temperature slides, and immediately refrozen by placing slides with sections into a slide box (VWR micro slide box #48444-003) with a single dessicant capsule (Humi-Cap see below). When the box is full, place the top on the box and store at -70°C. Sections cut and stored with dessicant are stable for in situ hybridization and immunohistochemistry for most antigens for over 5 years.

35S-RIBOPROBE SYNTHESIS FOR ISOTOPIC In Situ HYBRIDIZATION
by Josiah N. Wilcox, Ph.D. (based on Melton et al., 1984)

1. Pipet 12.5µl 35S-UTP (1200 Ci/mmol) into 1.5ml microfuge tube. Final concentration should be 12µM. Lyophilize in speed vac. (Do not use 35S-UTP that has been previously thawed. Order 250µCi vials and use up at first thawing or discard excess via radioactive disposal.)
2. To tube with the dried probe add:

   2.0µl 5x Transcription buffer
   1.0µl DTT, 100mM
   1.0µl RNasin
   1.0µl DNA (linearized plasmid 1µg/µl)
   2.0µl GTP+CTP+ATP Mix (stock solution containing 2.5mM @)
   2.0µl Sterile dH2O

   Mix thoroughly and centrifuge.
   Add 1.0µl RNA Polymerase (SP6, T7 or T3 as required)
   Mix gently by pipeting, do not vortex, and incubate 1-2 hours, 37°C.
   (Order all of the above as ready-made stocks from Promega Biotech).

3. To stop the reaction:

   Vortex
   Add 1.0µl RQ1 DNase to the transcription reaction above
   Incubate 15 min., 37°C.

   To extract RNA after DNase step, add to the reaction:
   20µl 1x TE
   1.0µl tRNA (50mg/ml)
   Vortex

4. Equilibrate one QuickSpin G-50 Sephadex column (Boehringer Mannheim Catalog No. 100-616) to room temperature for each riboprobe (15-30 min. at room temperature).

   Invert column gently for 20-25 times to suspend the gel.

   Remove TOP cap first, followed by the bottom cap.

   Allow the fluid within the column to drip through by gravity.

   Cut a collection tube (comes with column) approx. 5mm from bottom, place column in the collection tube and place the assembly in a 15ml tube.

   Spin for 2 min at 1100g or 2500rpm (setting #6 on IEC centrifuge).

   Discard fluid and collection tube.

5. Place the column in a new collection tube, add riboprobe to the CENTER of the column. Place the assembly gently into the 15ml tube and spin for 4 min at 1100g (#6 on the IEC). Remove the assembly gently with forceps, and measure the volume of riboprobe.
6. To monitor incorporation:

   Take 1µl from reaction and place in microfuge tube.
   Add 99µl 1xTE.
   Pipet 1.0µl of 1/100 dilution onto a small piece of DE81 ion exchange paper
   Wash 3 times 5 min. in 0.5M NaPO4, pH7.4
   Wash 10 seconds in dH2O
   Rinse briefly (<10sec) in 100% etoh
   Dry throughly and count in 10ml scintillation fluid

7. To the riboprobe add 1xTE to a final concentration of 300,000 CPM/µl. Use immediately for in situ hybridization or store 100µl aliquots at -70°;C up to one week in TE.
8. Just prior to starting the hybridization step:

   Thaw the riboprobe and keep on ice.

9. Make up Hybridization Mix as described in the in situ hybridization protocol

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)

	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)