F. Protocols and Reagents (continued)

6. Non-radioactive In Situ Using Biotin-labeled Riboprobes
7. Synthesis of Biotinylated Riboprobes
8. Sample Calculations for Non-radioactive In Situ
9. Solutions for Non-radioactive In Situ (biotinylated)
10. Non-radioactive In Situ Using Digoxigenin-labeled Riboprobes
11. Transcription of Digoxigenin-labeled Riboprobes

G. Sources of Material for In Situ
H. Acknowledgments

1. Calculate the amount(s) of stock RNA(s) needed for the Probe Working Solution (labeled-RNA in 2x Hybridization Buffer) in the hybridization experiment (see Calculations)

2. Prewarm 150ml of a 8µg/ml Proteinase K/PBS in a histological staining dish to 37°C for 40'

3. Prepare dehydration alcohols fresh (see step 9) with depc-H2O, in depc-treated staining dishes

4. Make up Probe Mix by combining:

25 µl Probe Working Solution (concentrated probe in 2xHyb buffer)

31 µl 20% Dextran Sulfate/Formamide

Denature for 10min at 95°C, chill on ice, then add

6 µl 200mM Vanadyl Ribonucleoside Complex (VRC)

_____

62 µl Total (of which only 50 µl is added to each slide

Vortex and keep the Probe Mix on ice until ready to hybridize slides

Note: All of the components of the Probe Mix, with the exception of the probe and VRC come with the BRL in situ kit (#18250-019). VRC is purchased from BRL (#15522-014) and diluted with sdH2O. See Calculations regarding preparing the Probe Working Solution which is a concentrated probe solution in 2xHyb buffer at 2.5x the desired concentration of probe in the actual hybridization.

5. Thaw slides to be hybridized (previously at -80°C) at 55°oC for 5'

6. Dip slides in a Proteinase/PBS solution, 8 µg/ml. Incubate at 37°C, 10'

(60 µl of prewarmed Proteinase K (20mg/ml) in 150 ml prewarmed 1x PBS)

7. Dip slides into 4% Paraformaldehyde/PBS at RT, 1'

8. Rinse slides briefly in 1x PBS, RT

9. Dehydrate slides in an alcohol series, 3' each:

50% EtOH (75ml 100% EtOH + 75ml depc-H2O)

70% EtOH (105ml 100% EtOH + 45ml depc-H2O)

90% EtOH (135ml 100% EtOH + 15ml depc-H2O)

100% EtOH (150ml 100% EtOH)

10. Air dry slides for approx 5' (though not preferrable, I have let these go as long as 45' without any obvious signal reduction)

11. Add 50 µl of chilled Probe Mix onto each air-dried slide

12. Incubate overnight at 55°C in humid chambers with filter paper that has been soaked in Box Buffer

13. Next day rinse slides in 0.2x SSC by dipping briefly 3 times (HOT WASTE)

14. Wash slides 2 times in 0.2x SSC at RT , 15' each (HOT WASTE)

15. Blot off SSC from slides with filter paper and place slides in humid chamber, this time with filter paper soaked in H2O. Don't let slides dry out before putting on blocking solution

16. Cover each tissue section with 100-300 µl of Blocking solution (BRL kit) and incubate at RT, 15'

17. During this time, make up the Streptavidin-Alkaline Phosphatase (SAP) Conjugate solution:

For each slide combine (multiply by the number of slides needed):

10 µl Streptavidin-Alkaline Phosphatase (BRL kit)

90 µl Conjugate dilution biffer (BRL kit)

_____

100µl Total

18. Remove blocking solution from slides by blotting with filter paper

19. Immediately after removing blocking solution, add 100 µl SAP conjugate solution to each tissue section and place back into humid chamber, incubating at RT, 15'

20. Wash slides 2 times in Tris Buffered Saline (TBS), at RT, 15'

21. Incubate slides in ASB (150 ml) with 1 ml Levamisole (30mg/ml; Final concentration = 200µg/ml), at RT , 5'

22. Add NBT and BCIP to fresh ASB that has been prewarmed to 37°C

150 ml prewarmed ASB

600 µl NBT (BRL kit)

498 µl BCIP (BRL kit)

NOTE: NBT and BCIP are shipped in the BRL kits at 4°C, but store at -20°C as reagents begin to degrade after 6 months.

23. Incubate at 37°C, 30' - check color development under microscope every 10' thereafter until color is satisfactory. Background may increase with increased color development time

24. Stop color development by rinsing slides in H2O

25. Air dry slides

26. The slides may be counterstained with any stain, however you may not dehydrate and clear the hybridized slides in xylene as the NBT/BCIP crystallizes reducing the signal intensity and/or increasing background. We have used a light Methyl Green counterstain which shows up well against the NBT/BCIP. The slides are rinsed in H2O after staining and are coverslipped with an aqueous medium such as Gelmount from Biomedia Corp. (available from Baxter). Coverslips should be sealed with fingernail polish to prevent the aqueous mounting medium from drying out.

This procedure uses the RNA labeling kit provided by GIBCO-BRL (Cat# 18093-013) and is performed essentially as described by the manufacturer except with the addition of a small amount of 35S which is used to monitor RNA synthesis.

1. Mix in centrifuge tube:

30.0 µl depc H2O

5.0 µl 10x Transcription Buffer

2.5µl 0.1 M DTT

5.0µl 10x rNTP mix (includes biotin-14-CTP)

1.0µl 35S-UTP (10µCi, >1000Ci/mmol)

1.0µl Linearized cDNA (1µg/µl)

2.5µl Placental RNase Inhibitor (10U/µl)

3.0µl RNA polymerase (40U per 50µl rxn)

________

50 µl Total

Note: The specific RNA polymerase you use will depend upon your plasmid and the orientation of your cDNA insert. All of the components above with the exception of the probe and 35S-UTP come with the BRL kit.

2. Incubate at 37°C for 1.5 hours

3. Add 2 µl depc-0.5 M Na2EDTA (pH 8.0) (BRL kit). New volume = 52 µl

(stops the transcription reaction)

4. Take out 1µl and place on small piece DE81 ion-exchange paper to determine total (incorporated + unincorporated nucleotides) counts. Dry paper briefly on parafilm on top of a 65°C heat block (this temp "doesn't have to be exact"). When dry, place paper into 10ml scintillation fluid and count)

5. Take an additional 1µl and place on DE81 paper. This time wash off unincorporated nucleotides with 0.5 M NaPO4. These counts are used as quality control to ensure that the transcription reaction proceeded as planned.

DE81 Washing: Rinse 3 times in 0.5 M NaPO4, 5' each

(Discard liquids in 35S waste disposal)

Rinse 1 time in H2O, 10 sec

Rinse 1 time in 100% EtOH, briefly

Dry again on parafilm on heat block

Place into 10ml scint fluid and count in beta-counter

 

New volume = 50 µl

6. Remove unincorporated nucleotides by sephadex G-50 spin columns as follows

The spin columns are obtained from Boehringer Mannheim (Cat# 100-616) and are used essentially as described by the manufacturer.

1. Allow column(s) to come to RT and invert several times to mix.

2. Remove top cap first, then bottom, and allow buffer to drip out.

3. Prespin column(s) at 1100 x g for 2' in a table-top centrifuge to remove packing buffer.

4. Add up to 50µl of RNA sample, slowly to the center to the column.

5. Centrifuge at 1110 x g for 4' and recover the eluate which is your purified RNA sample.

6. With a pipettor, take up the sample to determine the new volume.

(Final volume of 25-30µl from a 50µl sample is common).

7. Take 1µl from the Sephadex-purified sample and place onto DE81 ion-exchange paper to count incorporated nucleotides. Dry paper briefly on parafilm on top of a 65°C heat block (this temp "doesn't have to be exact"). When dry, place paper into 10ml scintillation fluid and count).

Note: You can check purity of probe at this point by counting washed and un-washed DE81 filters but it has been our experience that there are no free nucleotides after column purification.

8. Calculate amount of RNA available for hybridization from CPM (see Calculations). Typical yields from 1µg cDNA may range from 4-30µg.

9. Split final amounts of synthesized RNA into concentrated stock aliquots and store at -80°C. These stocks may then be thawed and diluted in 2x Hyb Buffer as needed for individual hybridization experiments.

Note: The concentration of the probe off the column is in the range of 300-1000ng/µl and the final concentration in the hybridization is on the order of 0.3ng/µl (approximately 7.8ng used per slide). It is possible to dilute this preparation in 1xTE and store a number of aliquots for use at a later time. We have no information regarding the stability of the probe but it may be stable for some months at -80°C.

A. Specific activity of trace isotope added to transcription:

(You will need this number to use in calculating the amount of RNA synthesized)

1) Concentration of cold UTP in transcription reaction:

1mM UTP (final concentration in 50µl rxion)

2) Moles of cold UTP in the 50 µl transcription reaction:

(1 x 10-3 moles/L)(50 x 10-6L) = 5 x 10-8 moles cold UTP

3) Number of Ci of 35S-UTP added to transcription reaction:

(Stock is 1000Ci/mmol; 10µCi/µl)

1 µl added of 10µCi/µl = 10µCi = 10x10-6Ci 35S-UTP added

4) Moles of 35S-UTP added to transcription reaction:

Stock is 1000Ci/mmol = 1000 x 103Ci/mol

Number of Ci 35S-UTP added = 10 x 10-6 Ci

Specific activity of 35S-UTP 1000 x 103 Ci/mol

= 1x10-11 mol 35S-UTP added to the transcription reaction

5) Total moles of UTP added to transcription reaction (cold + hot):

1.0 x 10-11 mol 35S-UTP

+ 5.0 x 10-8 mol cold-UTP

5.0001 x 10-8 mol

(35S-UTP does not contribute significantly to the amount of UTP in the reaction)

6) Specific Activity of 35S-UTP in the transcription reaction:

(10.0 x 10-6 Ci (step A3)) / (5.0 x 10-8 mol (step A5))= 200 Ci/mol in the 50 µl reaction

 

B. Estimation of RNA synthesis in biotinylated transcription reactions:

(you will need this number to calculate how much probe to apply to each tissue section)

1) Count 1µl of sample from step 7 (Sephadex column protocol). Assuming a

50µl total volume calculate the total CPM of incorporated nucleotides in the

sample:

________ CPM/µl x 50 µl = (X) CPM Total incorporated in 50 µl sample

2) Convert CPM to Ci of synthesized RNA:

(assuming 100% counting efficency DPM=CPM)

(X) CPM / (2.22 x 1012 DPM/Ci) = (Y) Ci UTP

Note: 1 Ci = 2.22 x 1012 DPM

3) Estimate moles UTP labeled(synthesized) in RNA:

(Y) Ci UTP (step B2) / (200 Ci/mol (step A6)) = (Z) moles UTP incorp in RNA

4) Calculate the number of moles of all NTP's in RNA:

(moles UTP x4, there are 4 NTPs and have only accounted for 1)

(Z) moles UTP x 4 = (Q) moles NTPs incorp in RNA

5) Amount of RNA synthesized:

(Q) moles NTPs x 330g/mol = (final answer) g RNA

Note: 330g/mol is the approximate molecular weight of an NTP

C. Calculation of amount of probe needed in Probe Mix:

The Probe Mix (for each slide to hybridize) consists of:

25 µl Probe Working Solution (concentrated probe in 2xHyb buffer)

31 µl 20% Dextran Sulfate/Formamide

Denature for 10min at 95°C, then add

6 µl 200mM Vanadyl Ribonucleoside Complex (VRC)

_____

62 µl Total (of which only 50 µl is added to each slide)

Note: The BRL in situ booklet (p. 18) suggests that the final concentration of labeled RNA be 0.1 µg/ml to 0.5 µg/ml in a 50µl hybridization reaction volume and that 2.5 times this desired concentration be dissolved in 2x Hybridization buffer to make the Probe Working Solution above. This does not work out to exactly 1xHyb buffer or 1x probe in the final hybridization but this is what the booklet suggests and is what we have been doing.

For example I found a final concentration of 0.125 µg/ml to yield a good signal with low background for rat POMC mRNA. So to make the Probe Mix above 25µl of a 0.3125µg/ml of the POMC Probe Working Solution in 2x Hyb Buffer would be added for each slide in the experiment. You may need to optimize this.

This works out to 7.81ng of biotinylated RNA for each slide in the experiment.

Tris-buffered Saline (TBS)		Final Conc
Trizma Base	12.10 g	100mM
Sodium Chloride	8.77 g	150mM
Add dH20 to 1 Liter and pH to 7.5 with 1N HCl.

Alkaline Substrate Buffer (ASB)		Final Conc
Trizma Base	12.10 g	100mM
Sodium Chloride	8.77 g	150mM
Magnesium Chloride	10.20 g	50mM
Add dH20 to 1 Liter and pH to 9.5 with 1N HCl.

Other solutions in main in situ hybridization protocol

Note: This is a preliminary protocol which may have problems with specificity and/or sensitivity which is under current investigation.

1. Take slides to be hybridized out of -80°C and thaw at 55°C, 5'

2. Quickly transfer slide to 4% Paraformaldehyde at 4°C, 10'

3. Wash twice in depc-treated 0.5x SSC, 5' each

4. Wipe off excess PBS from slides and add an 80µl drop of rHB2 (Hybridization Buffer from 35S-labeled riboprobes) onto to the tissue.

5. Incubate at 37°C in a humid chamber, with filter paper that has been soaked in Box Buffer (see solutions)

6. Prepare probe while slides are prehybridizing:

Spin down probe(s) at 14,000 RPM, 15'

Discard EtOH in HOT WASTE

Add 500µl 70% EtOH, vortex, and spin down again

Resuspend in TE to approximately 50ng/µl

Place 1µl onto DE81 paper to monitor incorporation:

Dry on parafilm

Wash DE81 paper in NaPO4, 3 times, 5' each

Wash 1 time in dH2O

Wash 1 time in 100% EtOH

Dry and count in 10 ml scintillation fluid

 

Based on counts, make up probe mix:

100ng probe + 1µl tRNA(50mg/ml) per slide

 

Denature probe at 90°C, 3'

Place on ice

Add 20µl ice cold rHB2 per slide

Vortex, hold on ice

7. Add 20µl probe directly to 80µl of rHB2 prehybridization solution on slide, and hybridize overnight at 55°C.

8. The following day, wash slides in 2x SSC, ßME, EDTA 1 hour, RT (HOT WASTE)

9. Wash in 1x SSC, ßME, EDTA twice for 30' at RT (HOT WASTE)

10. Wash in 0.5x SSC (100ml 20xSSC + 4000ml dH2O), 30', 55°C

11. Wash in 0.5x SSC, 30', RT

12. Wash in Buffer 1, 5', RT

13. Incubate slides in sheep-anti-digoxigenin-alkaline phosphatase (Boehringer Mannheim), diluted 1:500 in Buffer 1 + 1% BSA + 0.3% Triton X-100, overnight at 4°C, adding 100µl per slide.

example:

1.0µl Sheep

5.0µl 1mg/ml BSA (1% final concentration)

1.5µl Triton X-100

492.5µl Buffer 1

______

500.0µl Total (i.e. enough for 5 slides)

14. After antibody incubation, wash 3 times in Buffer 1, 15'

15. Wash in 100mM Tris, pH 8.2, 5'

16. Make up Vector Red substrate (Vector Laboratories cat#SK-5100) just prior to use:

5ml 100mM Tris, pH8.2

1 drop Levamisole

2 drops each of solutions 1, 2, 3 in the kit

17. After Tris wash (step 15), wipe off Tris and add 4-5 drops of substrate. Incubate at RT in the dark.

18. Check after 10', if brown precipitate present then shake off substrate, add fresh substrate and continue to incubate in the dark.

19. Continue checking color development under microscope every 10' and stop color development in water.

20. Counterstain with light hematoxylin, no eosin. Dehydrate in ethanols, clear in xylenes, and coverslip in any non-aqueous mounting medium.

NBT/BCIP staining if desired:

1. After antibody incubation, wash 2 times in Buffer 1, 15'

2. Wash in Buffer 3, 5'

3. Develop color in Buffer 3 + NBT + BCIP:

10ml Buffer 3

45µl NBT (75mg/ml in 70% DMSO)

35µl BCIP(50mg/ml in 100% DMSO)

Note: The NBT and BCIP can be made up as stocks and stored at -20°C

 

4. Stop color development in water.

5. Coverslip after staining with an aqueous mounting medium. Do not dehydrate and clear in xylenes.

1. Pipet 12.5µl 35S-UTP (1200 Ci/mmol) into eppendorf tube. Final concentration should be 12µM. Dry in speed vac. (Used to monitor RNA synthesis)

2. To tube of dried 35S, add:

5.0µl UTP/digUTP mix

10.0µl 10x transcription buffer (Promega)

5.0µl RNasin (Promega)

5.0µl linearized cDNA (1ug/ul)

10.0µl AGC nucleotide mix (@ 2.5mM each)

5.0µl DTT (Promega)

5.0µl sdH2O

5.0µl RNA polymerase

_____

50µl Total

Note: UTP/digUTP is made by adding: 6.5µl 10mM UTP + 3.5µl 10mM digoxigenin-11-UTP (Boehringer Mannheim #1209 256). Store small aliquots at -20°C.

3. Incubate at 37°C for 1- 1.5 hours.

4. Take out 1µl and place on small piece DE81 ion-exchange paper to determine total (incorporated + unincorporated nucleotides) scintillation counts. Dry paper briefly on parafilm on top of a 65°C heat block (this temp "doesn't have to be exact"). When dry, place paper into 10ml scintillation fluid and count.

5. Take an additional 1µl and place on DE81 paper to determine counts incorporated into RNA. Wash off unicorporated nucleotides with 0.5 M NaPO4:

Rinse 3 times in 0.5 M NaPO4, 5' each

(Discard liquids in HOT WASTE )

Rinse 1 time in H2O, 10 sec

Rinse 1 time in 100% EtOH, briefly

Dry again on parafilm on heat block

Place into 10ml scint fluid and count in scintillation counter

Note: Use these counts to monitor the quality of the transcription reaction.

6. Add 1.0µl RQ1 DNase (Promega) into the transcription reaction above and incubate 15' at 37°C.

7. Remove unincorporated nucleotides by Sephadex G-50 spin columns (Boehringer Mannheim Cat# 100-616) as described in previous section.

8. Determine the amount of RNA in purified preparation (see Calculations).

Note: Typical yields from 1µg cDNA may range from 4-30µg.

9. Aliquot labeled riboprobe and store at -80°C. These stocks may be thawed and diluted as needed for individual hybridization experiments. (Storage and stability unknown).

Buffer 1 (1 L) 100mM Tris, 150 mM NaCL
1M Tris-Cl pH 7.4	100 ml
1M NaCl	150 ml
dH20	750 ml

 

Buffer 3 (1 L) 100 mM Tris, 100 mM NaCl, 50 mM MgCl2
1M Tris-Cl pH 9.5	100 ml
1M NaCl	100 ml
1M MgCl2	20 ml
dH20	780 ml

rHB2 (described above in protocol for 35S-labeled riboprobes)

 

 

Source of Materials Used for In Situ Hybridization

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

Humi-Cap dessicant capsules (#245-2)

Baxter Scientific Products

Nalgene utility boxes (#L1995-4)

Miles stain dishes (#S7631-6)

Miles stain racks (#S7636)

O.C.T. (#M7148-4)

Tissue culture chamber slides (#T4135-4)

Peel-a-Way tissue molds (#M7275-3, #M7275-2)

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

Gills hematoxylin #2 (#4570)

Alcoholic Eosin Y 1% (#17269)

VWR

Micro slide boxes black (holds 25@) (#48444-003)

Slide grips (#48440-002)

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

Kodak NTB2 emulsion (#1654433)

Whatman

3MM paper (#3030M917)

DE81 filter paper (#3658M323)

Fisher Scientific

Autoimmerse heater (#L1995-4)

Superfrost/Plus Microscope Slides (#12-550-15)

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

Transcription buffer kits (ie. #P2490)

RNasin (#N2511)

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

35S-UTP (for riboprobe transcriptions) (#SJ1303 )

35S-dATP (low DTT concentration for tailing rxions) (#SJ1334)

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

RNase A (#R5125)

Proteinase K (#P4914)

tRNA (#R9001)

Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010 USA (415-697-3600)

Vectabond (#SP-1800)

Vector Substrate kit I (#SK-5100)

Acknowledgments:

I am deeply indebted to a number of people who have worked with me over the past 10 years who have each contributed to the development of this in situ protocol in some way including: Kathy Smith, Judy Hasko, Andrew Augustine, Hongzhi Xu and Romesh Subramanian. The non-radioactive protocols presented here were developed by Jose Rodriguez and Cynthia Sundell in my laboratory.

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