Vezina Group Protocols
IN SITU HYBRIDIZATION ON VIBRATING MICROTOME-CUT MOUSE TISSUE SECTIONS
Prior to starting
Prepare stock solutions, make DIG-labeled riboprobes, section mouse tissues with a vibrating microtome, make sample baskets and embryo powder as described below.
Make sample baskets
- 1. Use heated razor blade to cut off bottom of microcentrifuge tube (at or just below 100mL mark).
- 2. Cut polyester mesh into squares that are large enough to cover the cut part of the microfuge tube.
- 3. Heat cut edge of tube in flame until softened.
- 4. Press melted surface of microfuge tube firmly onto center of mesh square & hold for a few seconds to seal the mesh along entire circumference of tube.
- 5. Trim away excess mesh with scissors (may also heat near flame to melt down excess).
Make embryo powder
- 1. Collect mouse embryo tissue & store at –80°C until ready to make powder.
- 2. Place small amounts of frozen tissue into mortar, add liquid nitrogen & use pestle to grind tissue into powder (add more liquid nitrogen as needed).
- 3. Combine embryo powder with 4 volumes of acetone and homogenize with several strokes of dounce homogenizer until tissue is ground to a fine powder.
- 4. Transfer homogenate to a 15mL glass screw-top vial. Mix well and extract overnight at 4°C.
- 5. Pellet the embryo powder by centrifugation at 5000rpm for 10 min at 4°C.
- 6. Remove and discard supernatant.
- 7. Resupspend pellet in 4 volumes of fresh acetone and shake 2 hr at 4°C.
- 8. Pellet the embryo powder by centrifugation at 5000rpm for 10 min at 4°C.
- 9. Remove and discard supernatant.
- 10. Spread pellet onto #2 Whatman filter paper and air dry in fume hood.
- 11. Use mortar and pestle to grind dried pellet into fine powder.
- 12. Store powder in tightly sealed glass vial at 4°C.
DAY 1 – All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated.
- 1. Prep baskets
- A. Close basket cap, affix sticker to cap and label with probe name.
- B. Use heated 18 gauge needle to puncture two holes per basket cap (facilitates air movement).
- 2. Prepare supplies and preheat solutions.
- A. Transfer prehyb stock solution into sealed conical tube and preheat to 60.5°C.
- B. Fill a small plastic storage container (used as a hybridization chamber) with about ½ inch of tap water, cover container and preheat to 60.5°C.
- 3. Prep samples.
- A. Use spring scissors to remove most of the agarose from the vibrating microtome-cut tissue sections.
- B. Remove any debris if stuck to tissue sections.
- C. Add 2mL PBSTw and one basket to each well of a 24-well culture plate.
- D. Transfer sections into labeled baskets.
- 4. Incubate tissues for 30 min at 25°C in 2mL/well of 6% H2O2.
- 5. Wash tissues 4 x 5 min at 25°C with 2mL/well PBSTw.
- 6. Incubate tissues 12 min at 25°C in 2mL/well proteinase K solution.
- 7. Wash tissues 1 x 5 min at 25°C with 2mL/well PBSTw.
- 8. Incubate tissues 20 min at 25°C in 2mL/well post-fix solution.
- 9. Wash tissues 2 x 5 min at 25°C with 2mL/well PBSTw.
- 10. Prehybridization step: Incubate tissues inside hybridization chamber for at least 1 hr at 60.5°C in 2mL/well prehyb buffer.
- 11. Hybridization step: Add 0.65mg probe/well and incubate sections overnight in hybridization chamber at 60.5°C in probe+prehyb buffer.
- 12. Prepare and preheat solutions for Day 2: Add SDS to solution 1 (to a final concentration of 1%) and preheat overnight at 60.5°C.
DAY 2 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated.
- 1. Tissue sections are to remain in the baskets. Remove hyb buffer from each well and wash tissues 3 X 30 min at 60.5°C with 2mL/well Solution 1.
- 2. Prepare and preheat solutions.
- A. Prepare 50/50% mix of Solution1/Solution 2 and preheat to 60.5°C.
- B. Add RNase to Solution 2 and preheat to 37°C.
- C. Preheat one wash volume of Solution 3 to 25°C and two wash volumes to 60.5°C.
- 3. Wash tissues 1 X 10 min at 60.5°C with 2mL/well of 50/50% mixture of Solution 1/ Solution 2.
- 4. Wash tissues 4 X 10 min at 25°C with 2mL/well Solution 2.
- 5. Incubate tissues 15 min at 37°C in 2mL/well RNase solution.
- 6. Wash tissues 1 X 10 min at 25°C with 2mL/well Solution 2.
- 7. Wash tissues 1 X 10 min at 25°C with 2mL/well Solution 3.
- 8. Wash tissues 2 X 1 hr at 60.5°C with 2mL/well Solution 3.
- 9. Preheat Tissue Blocking (TB), Antibody Dilution (AD), and Antibody Absorption buffers to 25°C.
- 10. Wash tissues 3 X 10 min at 25°C with 2ml/well TBSTw.
- 11. Blocking step
- A. Incubate tissues at least 2 hr at 25°C in 2mL/well TB buffer.
- B. Add 3.3mL anti-DIG antibody per 600µL AA buffer & incubate at least 2 hr at 4°C.
- 12. Antibody step
- A. Centrifuge AA buffer containing antibody at 10,000 rpm for 1 min.
- B. Remove supernatant and add entire supernatant volume to 6mL AD buffer.
- C. In a humidified chamber, incubate tissues overnight at 4°C in 2mL AD buffer + antibody.
Day 3 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated.
- 1. Remove antibody solution and save.
- A. Add sodium azide to a final concentration of 0.2mM to prevent microbial growth.
- B. Store antibody solution at 4°C and reuse up to two additional times.
- 2. Wash tissues 8 X 10 min at 25°C with 2mL/well TBSTw containing 2 mM levamisole.
- 3. Remove tissues from baskets, use forceps to separate sections & remove visible debris, transfer tissues into clean microcentrifuge tubes.
- 4. Wash tissues 1 X 10 min at 25°C with 1mL NTMT containing 2 mM levamisole.
- 5. Detection step
- A. Protect tissues from light and incubate at 25°C in 1mL/tube of a 60/40% mixture of NTMT containing 2mM levamisole/BM Purple.
- B. Color development time ranges from several hours to several days. Change NTMT/BM Purple solution as needed (substrate will precipitate over time).
- Once color is fully developed, wash tissues 2 X 5 min at 25°C with 1mL/tube NTMT containing 2mM levamisole.
- 6. Bleaching step
- A. Post-fix tissues overnight at 4°C in 1mL/tube 4% PFA.
- B. Remove PFA and incubate tissues for 30 min at 25°C in 1mL/tube PBSTw containing 3% H2O2.
- C. Wash tissues 1 X 10 min at 25°C in 1mL/tube PBSTw.
- D. Store tissues at 4°C in 1mL/tube 4% PFA.
Solutions
Day 1
PBSTw: 1X PBS containing 0.1% Tween-20 and 0.2mM sodium azide. Pass through 0.2µm filter to remove insoluble material/contaminants.
6% H2O2: 1mL 30% H2O2 per 4mL PBS
Proteinase K: 0.25µL 20mg/mL proteinase K per 1mL PBSTw
Post-fix: 8µL 25% glutaraldehyde per 1mL 4% PFA
Prehybridization solution
| stock solution | final conc | for 1000mL |
| 100% formamide | 50% | 500mL |
| 20X SSC | 5X | 250mL |
| Blocking reagent | 1% | 10g |
| 10mg/mL yeast tRNA | 10µg/mL | 1mL |
| 10mg/mL heparin | 10µg/mL | 1mL |
| dH2O to vol. | -- | to 1000mL |
Aliquot 50mL volumes into conical tubes & store at –20°C.
Solution 1
| stock solution | final conc | for 500mL |
| 100% formamide | 50% | 250mL |
| 20X SSC | 5X | 125mL |
| dH2O | -- | 75mL |
| 10% SDS | 1% | 50mL |
Mix formamide, SSC & dH2O as shown above & store at –20°C.
DO NOT add SDS to freezer stock solution because SDS will precipitate in the cold.
Prior to using Solution 1 for ISH, add 1mL of 10% SDS per 9mL Solution 1 stock.
Day 2
Solution 2
| stock solution | final conc | for 500mL |
| 1M Tris-HCl, pH 7.5 | 10mM | 5mL |
| 5M NaCl | 0.5M | 50mL |
| 100% Tween-20 | 0.1% | 0.5mL |
| 0.2M sodium azide | 0.2mM | 0.5mL |
| dH2O | -- | 444mL |
Pass through 0.2µm filter to remove insolubles/contaminants, store at 25°C.
RNase: 5µL RNase (50µg/mL) per 1mL Solution 2
Solution 3
| stock solution | final conc | for 500mL |
| 20X SSC | 2X | 50mL |
| 100% formamide | 50% | 250mL |
| dH2O | -- | 200mL |
Store at –20°C
Tissue blocking (TB) buffer
| stock solution | final conc | for 500mL |
| 10X TBS | 1X | 50mL |
| 100% sheep serum | 10% | 50mL |
| 10% blocking reagent | 1% | 50mL |
| BSA | 1% | 0.5g |
| dH2O to vol. | -- | to 500mL |
| 100% Tween-20 | 0.1% | 0.5mL |
Mix TBS, serum, blocking reagent, BSA & dH2O as shown above.
Filter through #2 Whatman filters.
Add Tween-20.
Aliquot 6mL volumes into conical tubes & store at –20°C.
Antibody dilution (AD) buffer
| stock solution | final conc | for 500mL |
| 10X TBS | 1X | 50mL |
| 100% sheep serum | 5% | 25mL |
| 10% blocking reagent | 1% | 50mL |
| BSA | 1% | 0.5g |
| dH2O to vol. | -- | to 500mL |
| 100% Tween-20 | 0.1% | 0.5mL |
Mix TBS, serum, blocking reagent, BSA & dH2O as shown above.
Filter through #2 Whatman filters.
Add Tween-20.
Aliquot 6mL volumes into conical tubes & store at –20°C.
TBSTw: 1X TBS containing 0.1% Tween-20 and 0.2mM sodium azide. Pass through 0.2µm filter to remove insoluble material/contaminants.
Antibody absorption (AA) buffer
| stock solution | final conc | for 20mL |
| 1X TBSTw | -- | 17mL |
| 100% sheep serum | 5% | 1mL |
| 10% blocking reagent | 1% | 2mL |
| BSA | 1% | 0.2g |
| embryo powder | -- | 0.12g |
Shake at 4°C for 30 min to rehydrate embryo powder.
Aliquot 600µL volumes & store at –20°C.
Sheep serum (must be heat-inactivated before use).
To heat inactivate:
- thaw new bottle of serum
- incubate at 70°C for 30 min
- aliquot & store at –20°C
10% Blocking reagent
| stock solution | final conc | for 100mL |
| maleic acid | 100mM | 1.2g |
| 5M NaCl | 150mM | 3mL |
| dH2O to vol. | -- | to 100mL |
| Blocking reagent | 10% | 10g |
Mix maleic acid, NaCl & dH2O according to above, pH to 7.5 (note: strong buffer so difficult to pH, try using solid NaOH pellets to raise pH initially).
Add blocking reagent, microwave briefly to aid solubility (avoid boiling over, solution will be cloudy & viscous so watch carefully to ensure blocking reagent is completely dissolved in solution).
Aliquot 10 mL volumes into conical tubes & store at –20°C.
Day 3
2M Levamisole: Dissolve 4.82g levamisole in ~7mL double-distilled H2O (total volume should equal 10mL), aliquot 200µL volumes & store stocks at –20°C.
TBSTw + levamisole: 1X TBSTw containing 2mM levamisole.
NTMT + levamisole (inhibits endogenous alkaline phosphatases):
| stock solution | final conc | for 500mL |
| 1M Tris-HCl, pH 9.5 | 100mM | 50 mL |
| 5M NaCl | 100mM | 10mL |
| 1M MgCl2 | 50mM | 25mL |
| dH2O | -- | 415mL |
| 100% Tween-20 | 0.1% | 0.5 mL |
| 2M Levamisole | 2mM | 0.5mL |
Mix Tris, NaCl, MgCl2 & dH2O as shown above.
Pass through 0.2µm filter to remove insoluble material/contaminants. Store at 25°C.
DO NOT add Tween or levamisole to stock solution.
Prior to using NTMT for ISH, add 1µL of 100% Tween-20 and 1µL of 2M levamisole per 1mL NTMT stock.
3% H2O2: 1mL 30% H2O2 per 9mL PBSTw.
Abbreviations
BSA = bovine serum albumin
DIG = digoxigenin
H2O2 = hydrogen peroxide
PFA = paraformaldehyde
PBS = phosphate-buffered saline
PBSTw = 1X PBS + 0.1% Tween-20
SDS = sodium dodecyl sulfate (aka lauryl sulfate)
SSC = saline sodium citrate
TBS = Tris-buffered saline
TBSTw = 1X TBS + 0.1% Tween-20
Reagents and Supplies
Anti-DIG antibody, Fab fragments, cat # 11214667001, Roche
Blocking reagent, cat # 11096176001, Roche
BM Purple AP substrate, precipitating, cat # 11442074001, Roche
BSA, cat # BP1600-100, Fisher
Formamide, cat # F5786-1L, Sigma
Glutaraldehyde, 25% solution in H2O, cat # G6257-100ML, Sigma
Heparin, sodium salt, cat # H3393, Sigma
Hydrogen peroxide, 30% solution in H2O, cat # BP2633-500, Fisher
Levamisole, cat # L9756, Sigma
Magnesium chloride, cat # M33-500, Fisher
Maleic acid, cat # M0375-500G, Sigma
Paraformaldehyde, cat # 101176-014, VWR
PBS, w/out Ca & Mg, MP Biomedicals powdered media, cat # ICN1760420, Fisher
Proteinase K solution, 20mg/mL, biotechnology grade, cat # E195-5ML, Amresco
RNase, cat # R6513, Sigma
SDS, cat # S529-500, Fisher
Sheep serum, cat # S2263-500mL, Sigma
Sodium azide, granular, cat # S227I-100, Fisher
Sodium chloride, cat # BP358-212, Fisher
SSC, 20X solution, cat # S24022-4000.0, Research Products International
Tris-HCl, cat # BP153-1, Fisher
Tween-20, cat # BP337-100, Fisher
Yeast tRNA, cat # 109495, Roche
Polyester mesh, 33 micron, 12” x 24”, cat # CMY-0033-D, Small Parts Inc.
