In Situ Hybridization on Cryosectioned Tissue

Curators

Anthony S. Castanza, Ph.D., Department of Pathology, University of California San Diego email: acastanza@ucsd.edu

Abstract

This protocol is designed for In Situ Hybridization and colorimetric detection of digoxigenin-labeled RNA probes on paraformaldehyde fixed cryosectioned tissue. This protocol has primarily been tested on mouse embryos and on brain tissue and takes approximately three days.

Materials

Solutions

10X PBS (1 Liter)
82 g NaCl (FW=58.44)
2 g KCl (FW=74.55)
2 g KH₂PO₄ (FW 136.09)
11.5 g Na₂HPO₄ (FW=141.96)
Stir until solutes have dissolved, pH to 7.4 with HCl. Adjust volume to 1 Liter. AUTOCLAVE.

4% Paraformaldehyde in PBS (50 mL)
20 mL 10% PFA (made in water)
5 mL 10X PBS
25 mL autoclaved H₂O

1x PBS
100 mL 10X PBS +900 mL Autoclaved H₂O

20% Acetic Acid
10 mL Glacial Acetic Acid
40 mL Autoclaved H₂O

0.5M EDTA pH 8.0

1M Tris pH 9.5

1M Tris pH 8.0

1M Tris pH 7.5

0.1M Tris pH 7.5
10 mL 1M Tris pH 7.5
90 mL Autoclaved H₂O

2.3% Sodium (meta)Periodate (Make fresh on day of use)
1.15 g Sodium (meta)Periodate
50 mL Autoclaved H₂O

1% Sodium Borohydride DO NOT MAKE UNTIL IMMEDIATELY BEFORE USE
0.5 g Sodium Borohydride
50 mL 0.1M Tris pH 7.5
When ready for addition to staining jar: Add Borohydride to 200 mL Erlenmeyer flask. Add 50 mL 0.1M Tris. Swirl to dissolve Borohydride. The solution will vigorously react. Pour into Wheaton jar while still foaming.
Warning: The solution will bleach clothing.

Proteinase K (10mg/mL stock solution)
100 mg Proteinase K (Promega V3021)
500 uL 1M Tris (pH 8) (final 50mM Tris-HCl)
100 uL 1M CaCl₂ (final 10mM CaCl)
8 mL H₂O (Molecular Biology Grade)
Final Volume 10 mL. Make 1mL aliquots and store at -20C

Proteinase K Buffer (800 mL)
80 mL 1M Tris pH 8.0
80 mL 0.5M EDTA pH 8.0
Autoclaved H₂O to 800 mL

10X Salt Solution
23.38g NaCl (2M)
2.808g Tris HCl (0.089M)
0.265g Tris Base (0.011M)
1.38g Sodium Phosphate Monobasic Monohydrate (NaH₂PO₄.H₂O, 0.05M)
1.42g Disodium Phosphate (Na₂HPO₄, 0.05M)
20 mL 0.5M EDTA (0.05M)
Autoclaved H₂O to 200mL

10X TBS pH 7.5
60.55g Tris Base (FW: 121.1)
87.66g NaCl (FW: 58.44)
750 mL miliqH₂O
Stir until dissolved, pH to 7.5 with HCL (aprox. 30mL), bring to 1 Liter final volume with miliqH₂O

Prehybridization Solution
50 mL Formamide
10 mL 10X Salt Solution
40 mL Autoclaved H₂O

Hybridization Mix (Make in advance and store at -20)
1mL 10X Salt Solution
5mL Deionized Formamide
2mL Dextran Sulfate 50% Stock (made in H2O)
200uL 50X Denhardt's
200mg Roche Blocking Reagent
5uL Tween 20
1mL 10mg/mL Yeast tRNA
Autoclaved H₂O to 10 mL
Incubate at 60C up to 24 hrs with agitation to ensure all components are in solution.

Blocking Solution
1mL 10X Roche Blocking Solution
1mL Normal Goat Serum
1mL 10X TBS pH 7.5
10uL Tween 20
6990 mL H₂O (Molecular Biology Grade)

1X NTMT (made fresh; 250 mL for washes + color reaction
5mL 5M NaCl (100 mM)
25mL 1M Tris pH9.5 (100mM)
12.5mL 1M MgCl₂ (50mM)
207.5 mL Autoclaved H₂O

Reagents

NaCl [1]
KCl [2]
KH₂PO₄ [3]
Tris HCl [4]
Tris Base [5]
NaH₂PO₄.H₂O [6]
Na₂HPO₄ [7]
EDTA
Formamide (Molecular Biology Grade) [8]
CaCl₂
MgCl₂
Proteinase K [9]
PFA
Yeast tRNA (Roche)
Roche Blocking Reagent [10]
Goat Serum [11]
Tween 20 [12]
50X Denhardt's Solution [13]
Sodum (meta)Periodate [14]
Glacial Acetic Acid [15]
Sodium Borohydride [16]
Dextran Sulfate 50% Solution [17]
(-)-Tetramisole Hydrochloride [18]
Anti-Digoxigenin-AP Fab Fragments from Sheep [19]
NBT/BCIP [20]
Pap Pen [21]
200 Proof Ethanol
Xylene (or Substitute)
Permount

Equipment

Slide Moisture Chamber (Black) [22]
Wheaton Staining Jar [23]
Glass Coverslips
Incubators capable of 37-65 Celsius range.
Shaker/Rocker Table

Procedure

Day 0

• Pick slides and dry at room temperature for at least 30 minutes
• Fix slides in 4% PFA on a rocker/shaker table (Best result: overnight at 4C, 1 hour at Room Temperature is acceptable)

Day 1: Prehybridization and Hybridization

• Prepare "day of" solutions.
  • Prepare 2.3% Sodium (meta)Periodate
  • Prehybridization Solution (Make and pre-warm to 60C)
  • 0.1M Tris pH 7.5 (prepare for both wash and borohydride - do not add borohydride yet)
  • Prewarm Proteinase K buffer to 37C
  • 20% Acetic Acid

1) Rinse 3X briefly in Autoclaved Water (quick rinse, seconds, no timer).

• • Save 4% PFA refrigerated for step 11

2) Incubate in 2.3% Sodium (meta)Periodate solution for 5 minutes at room temperature on a rocker table.
3) Rinse 3X briefly in autoclaved water.
4) Incubate in 20% Acetic Acid for 5 minutes at room temperature on a rocker table.
5) Rinse 3X briefly in autoclaved water.
6) Rinse 1X in 0.1M Tris pH 7.5.

• • Prepare Sodium borohydride solution. See recipe note.
    

7) Incubate in 1% Sodium borohydride (dissolved in 0.1M Tris pH 7.5) for 5 minutes.
8) Rinse 4X briefly in autoclaved water.

• • Prepare Proteinase K solution by diluting 10 uL Proteinase K stock solution (10mg/mL stored at -20C) in 50mL Proteinase K Buffer (prewarmed at 37C). Shake to mix.

9) Incubate in Proteinase K for 5 minutes at 37C.
10) Rinse 3X in PBS (preferred) or autoclaved water.
11) Incubate in 4% PFA (saved from initial fixation step) for 5 minutes at room temperature.
12) Wash 3X briefly in autoclaved water.
13) Incubate 2X in prehybridization solution for 5 minutes at 60C

• • Incubate in solution once, pour out, incubate again. Save waste for filling Slide Moisture Chamber.
  • During this step, prepare the probe. Pre-warm hybridization mix to 60C. Dilute Dig-labeled RNA to 1ng/uL in hybridization mix. Prepare 100-150 uL per slide. Make sure solution is evenly mixed.

14) Remove slide from the jar, tap edge on a paper towel to gently dry. Add ~100uL probe in hybridization mix per slide.

• • Hybridization mix can be added dropwise directly on to each section.
  • Work quickly so that the sections do not dry out entirely.

15) Cover gently with glass coverslips.

• • Carefully place coverslips to avoid air bubbles. Do not press out bubbles/excess mix.
  • No excess hybridization mix should escape from the sandwich.

16) Fill the bottom of a Slide Moisture Chamber with waste prehybridization mix (or 50% formamide in 1X Salt Solution). 17) Place slides in the chamber carefully to not disturb coverslips.
18) Seal chamber in a plastic bag and incubate overnight at 60C.

Day 2: Post Hybridization

• Prepare "day of" solutions.
  • 100 mL 50% formamide in 1x salt solution (this is the same as prehybridization solution - make fresh for day 2). Warm to 60C before use.
    
  • 100 mL 0.5X Salt Solution (5 mL 10X Salt Solution + 95 mL Autoclaved water). Warm to 60C.

1) Carefully remove coverslips

• • Float off in prehybridization solution, or very gently lift off from one corner.
  • Transfer slides back to Wheaton jar.

2) Wash 2X 1 hour in 50% formamide in 1x salt solution (2 hours total) at 60C

• • Save formamide waste for next time.

3) Wash 2X 1 hour in 0.5X Salt Solution at 65C (2 hours total)

• • Use this time to prepare blocking solution. Store at 4C until ready. May be stored for up to 1 week.

5) Wash 2X 15 minutes in 1X TBS pH 7.5 at room temperature.
6) Block for 1 hour at room temperature in blocking solution.

• • Fill bottom of slide chamber with dH2O
  • Tap edge of slide of paper towel to dry
  • outline sections with pap pen
  • add 40-50 uL blocking solution per section

7) Dilute anti-Dig-AP antibody in blocking solution

• • 1:1000 for overnight incubation (1:2000 for over a weekend)
  • prepare ~50 uL per section

8) Remove blocking solution, add antibody diluted in blocking solution. Store at 4C in the chamber with water at the bottom.

Day 3

• Prepare NTMT Solution (200 mL)
  • Dissolve 25 mg of tetramisole hydrochloride in 50 mL of the NTMT

1) Transfer slides back to Wheaton Jar taping off Antibody solution.
2) Wash 6X 30 Minutes in 1X TBS pH 7.5 at room temperature on a shaker table.
3) Wash 15 minutes in NTMT with tetramisole.
4) Wash 2X 15 minutes in NTMT without tetramisole

• • Right before the second wash is finished, dilute 500 uL NBT/BCIP solution in 49.5 mL NTMT

5) Pour out the 2nd wash. Add NTMT with NBT/BCIP.

• • Cover with parafilm and wrap Wheaton jar in foil to protect from light.

6) Monitor Color reaction.

• • Checking at 10 minutes, 30 minutes, 1 hr, 2hr etc until color develops.
  • Some low abundance probes take a long time to develop. These may need to be left overnight at 4C or at Room Temperature. This step varies greatly and will have to be tested for your individual probe.

4) Stop reaction by washing 2X 5 minutes in 1X TBS

• • If performing subsequent immunodetection, wash in 1X PBS instead and skip following steps.

5) Dehydrate through Ethanol, Xylene series.

• • Water, Water, 75% Ethanol, 95% Ethanol, 100% Ethanol, 100% Ethanol, Xylene (or Substitute), Xylene (or Substitute), Xylene (or Substitute).
  • Dip slides briefly 10 times in each step of the series.

6) Coverslip with Permount.

Notes

Always run a Sense probe in parallel with the Antisense probe to ensure specificity of the reaction (at least the first time for any tissue/probe set).
All Day 1 and Day 2 procedures should (ideally) be performed in RNase-free conditions.
Unlike many NBT/BCIP reagents, this color product is stable through the ethanol/xylene dehydration and mounting procedure. However, it may fade after several months of storage and may exhibit some crystaline precipitation that is not desired.

Acknowledgments

This protocol was adapted from those provided by the Rubenstein lab at UCSF and the Grove lab (Chicago), and modified by F. Bedogni and G. Elsen (Hevner Lab, University of Washington)

References

Elsen GE, Hodge RD, Bedogni F, et al. The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map. Proc Natl Acad Sci USA. 2013;110(10):4081-6.
Bedogni F, Hodge RD, Elsen GE, et al. Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex. Proc Natl Acad Sci USA. 2010;107(29):13129-34.

Discussion

You can discuss this protocol.