insitu prtcl

IN-SITU HYBRIDIZATION PROTOCOL

DIGOXIGENIN AND FLUORESCEIN LABELED cRNA PROBES

TISSUE PREPARATION-CRYOSTAT SECTIONS

1) Perfused tissue: Anesthetized animals are perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer.

2) Section tissue utilizing a cryostat at -15° to -20° C. Sections are collected onto room temperature Superfrost slide. Store slides at -20° C until ready to pretreat and hybridize.

RESTRICTION DIGESTION

It is necessary to linearize the supercoiled plasmid (for plasmid transfection and purification see Appendix A) prior to transcription. A quantitiy of about 3-4 ug of total plasmid with insert should be suitable for 2-3 transcriptions. Determine the restriction enzyme to linearize with from the plasmid and insert maps.

Add the following into a microcentrifuge tube in the order shown

ITEM	AMOUNT
H2O	about 11 ul
Plasmid	2-3 ug, usually 4 ul depending on concentration
10x Restriction Enzyme Buffer	2 ul
Spermidine (80 mM) (optional)	1 ul
Restriction enzyme	1-1.5 ul
BSA 2mg/ml	1 ul
Total Volume	about 20 ul

Mix and spin briefly. Digest at 37° C for 2 to 16 hours, remove to ice and spin briefly. Ethanol precipitate DNA by adding a half volume of 7.5 M Ammonium Acetate and 2 volumes of 100% Ethanol, mix well, spin briefly and store on dry ice for about 20 minutes or at least -20° C overnight. Spin linearized cDNA at 12000-14000 rpm in a microcentrifuge for about 15’, remove solution, being careful to not disturb pellet. Wash pellet with 100 µl of 70% ethanol and respin for 5 minutes at 12000-14000 rpm. Remove 70% carefully and allow pellet to air dry for about 5 minutes or until slightly dry.

Resuspend pellet of cDNA in 4-6ul of TE (10mM Tris and 1mM EDTA, pH 8.0).

A portion, about 0.5 ul, of the resuspended, linearized samples should be run on a 1% TAE gel (see Appendix c Etbr Gel) to determine the completeness of the linearization. See Appendix B. If the linearization is incomplete, 1 ul of spermidine may be added to the next linearization reaction to assisist in enzyme activity.

PROBE SYNTHESIS

Combine the following nucleotides to make a nucleotide mix:

NTP mix made up and stored at - 20°C for multiple transcriptions

10.0 µl 10mM ATP (final conc. 500 uM) (2.5mM in mix)

10.0 µl 10mM GTP

10.0 µl 10mM CTP

6.5 µl 10mM UTP

3.5 µl 10 mM UTP-Digoxigenin or Flourescein

Combine the following in a microcentrifuge tube

3-4.0 µl NTP Mix

6.0 µl DH2O

1.0 µl cDNA (about 1 µg) previously linearized and checked on TAE gel

4.0 µl 5x Transcription Buffer

1.0 µl RNAsin

1.0 µl RNA Polymerase for promoter of interest

1 ul 2mg/ml BSA

1 ul 20mM DTT

The final volume should be limited to not more than 23 µl total.

Incubate the microcentrifuge tube at 37° C for SP6 Polymerase and 42°C for T7 and T3 polymerases, for 1 hour. An additional hour of incubation and/or another 1ul of polymerase may result in higher yields. If adding an additional 1 ul of polymerase, add it after 1 hour of incubation, then incubate an additional hour.

DNASE DIGESTION

Remove microcentrifuge tube from water bath and add

1.0 µl RNAse free DNAse (Promega)

.7 µl RNAsin (Promega)

1.0 µl Glycogen (Roche)

incubate for 15 minutes at 37° C. Remove tube to ice, spin down briefly. If continuing on to alkaline hydrolysis, bring volume up to 50 ul in each tube with DEPC dH2O. If alkaline hydrolysis is not necessary, skip to ethanol precipitation.

ALKALINE HYDROLYSIS (optional)

The alkaline hydrolysis step is performed only for probes more than 600 bp in length. It may assist in probe penetration in certain tissue types. A calculation is performed to determine an incubation time allowing for random cleavages resulting in a probe of the desired length of about 300 bp.

Li - Lf

t=___________ where t = time in minutes

k x Li x Lf Li = initial length Kb

Lf = final length (usually about .300 Kb)

k = constant .11 cuts/Kb/min\

Make up fresh 0.2M Na2CO3 (sodium carbonate; for 1 ml use 21.2 mg) and 0.2M NaHCO3 (sodium bicarbonate; for 1 ml use 16.8 mg). To alkaline hydrolize probe suspended in 50 ul of dH2O, add 30µl of sodium carbonate and 20 µl of sodium bicarbonate to the cRNA probe, incubate at 60° C for the appropriate amount of time calculated above.

Remove the vial to ice, spin briefly, and stop reaction by adding

3 µl 3M NaAc, pH 6.0 (Sodium Acetate)

5 µl 10% Glacial Acetic Acid

ETHANOL PRECIPITATION

Ethanol precipitate probe with 1/10 µl of 4 M LiCl and three times volume cold 100% ethanol.

Precipitate at -20 C (or colder) overnight or on dry ice for at least 30 minutes.

RESUSPENSION

Spin probe at 12000-14000 rpm in a microcentrifuge for about 15’ at (at 4⁰C if possible), remove solution, being careful not to disturb pellet. Wash pellet with 100 µl of 70% ethanol and respin for 5 minutes at 12000-14000 rpm. Remove 70% carefully and allow pellet to air dry for about 5 minutes or until slightly dry. If too dry resuspension becomes laborious.

Resuspend pellet of cRNA in 50-100 ul of sterile depc dH2O, tap well and spin. Repeat to assure resuspension. (Dot Blot see Appendix B)

PRETREATMENT

The slides selected for hybridization are treated as follows:

1) Slide boxes containing slides of interest removed from -20C freezer, labeled and placed into 4% Paraformaldehyde/1XPBS for 5 minutes

2) Rinsed in 1X PBST (0.1% Tween 20) for 3 x 10 minutes

3) Optional: To inhibit endogenous peroxidase incubate tissue 10 minutes in 0.3 to 3% H₂O₂ in PBST, then wash 3 x 5 minutes in PBST. The concentration of H₂O₂ is dependent on the amount of blood in the tissue and age of the H₂O₂ stock being used. Note that this step is only necessary if a peroxidase enzymatic reaction is to be used for coloration.

4) Proteinase K Digestion 10 -20 µg / ml in PBST (or 0.05 M Tris pH 7.5) for 10-20 minutes at room temperature or at 37 degrees celcius.

The concentration and time can vary depending on tissue type and enzyme activity.

5) Rinse 2 x 10 minutes in PBST to stop reaction.

6) Fix tissue 1 x 5 minutes in 4% paraformaldehyde/PBS

7) Rinse 2 x 10’ in 1 x PBST.

8) Brief (10”) DEPC H2O.

Slides may be left to dry at 37° C for up to 30 minutes at this step.
Prehybridization Mixture:

Prepare the following mixture. It can be aliquoted and frozen at -20° C for future use.

Stock Solution	Quantity	Final Concentration
100% Deion. Formamide -20C	4.500 ml	50% Deion. Formamide
20X Hybridization Salts Stock RT (see appndx)	2.250 ml	5X Hybridization Stock
100X Denhardts -20C (see appndx)	0.090 ml	1X Denhardts
10% Tween 20 or 10% SDS both Stocks @ RT	0.180 ml	.2% Tween or SDS
DEPC dH2O	0.225 ml

Volume 7.245 ml (80.5% of 9.0 ml Final volume)

PREHYBRIDIZATION BUFFER:

Prehybridization reduces non-specific hybridization. In some instances, it is not necessary.

To make 2.0 ml of prehybridization buffer combine the following into a microcentrifuge tube.

Stock Solution

Quantity

Final Concentration

Salmon Sperm DNA

10 mg/ml

50 µl

250 µg/ml

Poly A

10 mg/ml

50 µl

250 µg/ml

DEPC Treated

H2O

290 µl

Total 390 µl

Combine well by pipetting up and down the mixture with the tube, seal tube well and place into boiling water bath for 3’, quench on ice.

Remove 1.610 ml of Prehybridization Mixture to a suitable vial and add the 390 µl of ss DNA, Poly A and DEPC H2O solution, mix well. This solution is now at final concentrations for each of the constituents.

Place a suitable amount into a microcentrifuge tube to completely cover tissue.

or approximately 100 µl per slide, coverslipped with parafilm or glass, placed into a formamide humidified chamber.

Prehybridize at 37-42 degrees Celcius for 1-2 hours.

HYBRIDIZATION BUFFER:

Prepare the following mixture. It can be aliquoted and frozen at -20° C for future use.

Stock Solution	Quantity	Final Concentration
100% Deion. Formide.-20C (see appndx)	4.500 ml	50% Deion. Formamide
20X Hybridization Salts Stock RT(see appndx)	2.250 ml	5X Hybridization Stock
100X Denhardts -20C (see appndx)	0.090 ml	1X Denhardts
10% Tween 20 or 10% SDS both Stocks @ RT	0.180 ml	.2% Tween or SDS
50% Dextran Sulphate	0.900 ml	5% Dextran
DEPC dH2O	0.225 ml

Total Volume 8.145 ml (90.5% of 9.0 ml Final volume)

Using theoretical, gel estimations, or dot blot estimations for the amount of probe transcribed with the digoxigenein reaction, estimate a quantity for a concentration of 0.5 ng-.2ng / µl. For most of the digoxigenin and flourescein transcriptions performed, dilutions of 1:40 - 1:100 are typical.

For tissue coverage, about 100 ul per slide is necessary. Wholemount coverage in a microcentrifuge tube varies from as little as 40 ul to 200 ul. Calculate the amount of probe at the above concentration, ss DNA at 250 µg/ml, Poly A at 250ug/ml and water, if necessary, to make up 9.5% of the final volume desired. This quantity should be combined into a microcentrifuge tube, boiled for 3’ and quenched on ice. Then add the appropriate volume of hybridization buffer for the quantity desired.

Example:

Hybridizing 20 slides, each slide needs 100ul and make a little extra so there is no shortage. Therefore, 21 x 100ul = 2.1 ml.

The Hybridization Buffer is 9.5% short of final volume to allow for the ssDNA, Poly A and Probe. Therefore, .095 (9.5%) of 2100 = 199.5ul

52.5 ul of 10 mg/ml Poly A for a final concentration of 250 ug/ml

52.5 ul of 10 mg/ml ss DNA for a final concentration of 250 ug/ml

21.0 ul of probe for a 1:100 dilution.

126.0 ul Net Volume

199.5 ul -126.0 ul = 73.5 ul volume of water necessary

73.5 ul ddH2O

Combine and boil the above (Poly A, ssDNA, probe and water) for 3 minutes and quench on ice. Add 1900.5 ul of hybridization buffer for a final volume of 2100 ul.

Apply solution to slides and coverslip with parafilm or cover wholemount tissue in a microcenrifuge tube. Hybridization temperatures are between 55 and 65 ° Celcius,

WASHING

From a stock of 20x SSC (see appendix) each of the solutions are made

Place tissue into a solution of 2x SSC at 60 degrees Celcius for 3 x 10 minutes

Place tissue into a solution of 0.2x SSC at 60 degrees Celcius for 3 x 10 minutes

Place tissue into a solution of 50% .2x SSC and 50% 0.1MPBST (see appendix) at room temperature for 1 x 10 minutes.

At this stage all buffered solutions used are 0.1 Molar Phosphate buffer with saline with 0.1% Tween-20 (0.1M PBST).

Place tissue into a solution of 0.1MPBST for 2 x 10’.

IMMUNOHISTOCHEMICAL DETECTION OF DIGOXIGENIN OR FLOURESCEIN

To reduce non-specific binding, incubate tissue with 1% Normal serum, 2 mg/ ml BSA in 0.1M PBS with 0.3% Trition x-100 at room temperature for 1-2 hours.

For fluorescent detection, incubate tissue in biotinylated mouse anti-digoxin (Jackson) overnight at 4⁰C. Wash with 0.1M PBST 3 x 10 min. Incubate in ABC (Vector) 1-2 hours. Wash 3 x 10 min. with 0.1M PBST. Incubate in TSA A488 or A568 (Molecular Probes) for 3 to 30 minutes. See Product info from TSA kit for complete details. Monitor under microscope to determine the optimum time. Wash thoroughly in 0.1M PBST, i.e., 4 x 10 min then overnight wash.

(Alternatively, fluorescent detection can be accomplished with a more direct method using peroxidase conjugated anti-digoxigenin (Roche), followed by TSA. This method is typically not as sensitive as the ABC method.)

For non-fluorescent detection, incubate tissue in anti-digoxigenin Fab-fragments-alkaline Phosphatase labeled 1:1000 (Roche) diluted in 1% Normal serum, 2 mg/ ml BSA in 0.1M PBS with 0.3% Trition x-100 at room temperature for 2 hours or overnight at 4⁰C. Continue with Immunohistochemistry washes below.
IMMUNOHISTOCHEMISTRY WASHES

For alkaline phosphatase reaction products.

Wash 3 x 10 minutes in .1 MPB at room temperature.

Rinse briefly with dH2O then wash 2 x 5 minutes in Coloration Buffer.

Coloration Buffer For 100 mLs

100 mM Tris pH 9.5 10 mL of 1 M Tris

50 mM MgCl2 1.01 gram of MgCl2 w 6H2O

100 mM NaCl 2 mL of 5 M NaCl

0.1% Tween 20 100 µl Tween 20

Sterile DH2O to 100 ml

check that pH is 9.5

COLORATION

For Blue Precipitate

Just before use, combine 45 µl of NBT stock (Beohringer-Mannheim) with 10 mL coloration buffer then add 35 µl of BCIP stock (Boehringer-Mannheim), mix well.

For Red Precipitate

Take 1 fast red tablet from Boehringer’s Multi color Detection Kit and dissolve into coloration buffer, then filter using standard filterpaper.

Using tissue culture plasticware, provide enough solution to cover tissue and allow coloration to occur in the dark for 15 minutes up to 2 hours at room temperature. Note that this chromagen is also visible using rhodamine cubes in standard flourescent microscopes

If longer coloration times are required, moves tissue and or slides to a 4 degrees Celcius environment for overnight coloration.

WASH

After coloration is satisfactory, wash sections 3 x 10’ in 0.1 M PBS.

The tissue can be refixed in 4% Paraformaldehyde/PBS for storage.

Tissue on slides can be coverslipped in the usual manner without dehydration. Ethanols have been reported to precipitate the color product, however in practicality a quick dehydration to xylene and coversliping with permount does not seem to result in any loss of color for the NBT/BCIP precipitate. The fast red is susceptible to fading in organics.

Stock Solutions