J. Rudnick 11/08

In Situ Hybridization Protocol Detection in FFPE Tissues modified BD

Use only DPEC water as a solvent (referred to just as “DPEC” in this protocol). Rinse all previously autoclaved glassware with DPEC. Rinse plastic histology containers (ISH use only) with milli-q water and then again with RNAse Zap, rinse again with milli-q. Use DNAse/RNAse free tips! Everything sterile!

  • Essential Reagents to Purchase

LNA detection probes for Exiqon 5’ label DIG

1. U6 99002-01 $225 Tm: 75

2. Scramble 99004-01 $225 Tm: 78

3. Hsa-mir-24: 18121-01 $375 Tm: 80

4. Hsa-mir-221: 18115-01 $375 Tm: 80

Anti-DIG/HRP antibody (mouse mono-clonal, clone 21HB)

Novus: NB100-41330

Abcam: ab6212

TSA kit #2 with anti mouse Alexa Flour 488 tyramide Invitrogen #T20912 $399

Acetic Anhydride Sigma A6404-200ml $22.80

50X Denhards solution Sigma D-2532-5ml $73.80

Blocking reagent Roche 11096176001 $110

Protease K Roche 25mg 03115836001 $42

Prolong Gold with Dapi Invitrogen P36931 $128

Probes

Hybridization temp: J: 22-25 below calculated tm: for this set, use 55 degrees

Probes come at 25uM concentration

Dilute 5ul probe + 45ul RNAse free water to give 2.5uM. Store remaining un-diluted probes in -80

Aliquot diluted probe and store at -80.

  • Buffers
  • 8 L of DPEC treated water (“DPEC”). YES, 8 L! YOU WILL USE ALL OF IT.

For every 1 L milli-q, add 1 mL DPEC

Shake vigorously

Let sit O/N in fume hood

Autoclave to remove DPEC, 1 hr

If soln smells very sweet post autoclaving/cooling, let sit longer in fume hood with cap loosened to evaporate the odor.

  • 1L of 1 M Tris-HCl, pH 7.5 DO NOT NEED TO MAKE THIS MUCH!!!

121.1 g Tris base

~65 mL 37% HCl

pH to 7.5

Bring to 1L volume with DPEC

Autoclave

  • 500 mL of 5 M NaCl

146.1 g NaCl

500 mL DPEC

Autoclave

  • 1L of 20X Sodium Citrate (SSC)

175.3 g NaCl

88.2 g sodium citrate

pH to 7.0 with 1 M HCl (few drops)

Adjust volume to 1 L with DPEC water

Autoclave

For 500 mL (each) of SSC Dilution Series:

2X SSC: 50 mL of 20X SSC, 450 mL DPEC, Autoclave

1X SSC: 25 mL of 20X SSC, 475 mL DPEC, Autoclave

0.5X SSC: 12.5 mL of 20X SSC, 487.5 mL DPEC, Autoclave

0.2X SSC: 5 mL of 20X SSC, 495 mL DPEC, Autoclave

  • 500 mL (each) of Ethanol Rehydration Series Solvents

For 99% : 5 mL DPEC, 495 mL 100% EtOH

For 96% : 20 mL DPEC, 480 mL 100% EtOH

For 90% :50 mL DPEC, 450 mL of 100% EtOH

For 70% : 150 mL DPEC, 350 mL of 100% EtOH

For 50% : 250 mL DPEC, 250 mL 100% EtOH

For 25% : 375 mL DPEC, 125 mL 100% EtOH

  • 4% Paraformaldehyde (PFA)

10 mL aliquot of 16% PFA vial

30 mL 1X PBS (diluted from 10X stock in DPEC H20)

Prepare in a sterile 50 mL falcon tube, use within 1 month

Make fresh every time (or use within 3days)

Add 1g paraformaldehyde powder to 25ml PBS add 2.5ul 10N NaOH

Heat until dissolved

  • 200 mL Proteinase K Buffer (pre-warmed to 37 C before using)

187.4 mL DPEC H20

1.8 mL 1 M Tris-HCl ([final] is 10 mM Tris-HCl, pH 8)

9 mL 100 mM EDTA ([final] is 5 mM EDTA, pH 8)

1.8 mL 5 M NaCl ([final] is 50 mM NaCl)

  • 10 mL Hybridization Soln: based on silahtaroglu et al. (see obernosterer for alternative)

5 mL formamide ([final] is 50%)

2.5 mL 20X SSC ([final] is 5X)

120 L 25 mg/mL yeast tRNA ([final] is 300 g/mL)

200 L of 50X Denhardt’s Soln ([final] is 1X)

50L 20% Tween-20 ([final] is 0.1%)

2.13 mL DPEC H20

Divide into 150 ul aliquots and store -20 C.

This hybridization buffer has a pH of ~6-6.5

  • 200 mL 3% H2O2 in PBS

(prepared fresh)

18 mL 30% H2O2 ([final] is 3%)

182 mL 1X PBS (diluted from 10X stock with DPEC)

  • 200 mL 0.2 % Glycine Soln (w/v)

0.48 g glycine

400 L glycerol

200 mL 1X PBS (diluted from 10X stock with DPEC)

  • Acetylation Soln

(prepared just before use in fume hood; all pipets to be discarded in solid waste container)

12 mL of 1 M HCl ([final] is 66 mM)

2.68 mL triethanolamine ([final] is 1.5% v/v)

180 mL DPEC

Just before dipping the slides, add 1.2 mL acetic anhydride ([final] is 0.66% v/v)

YOU MUST ADD ACETIC ANHYDRIDE LAST! IT VIOLENTLY REACTS WITH PURE WATER!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

  • 200 mL 0.5 % Triton X 100

1 mL Triton X 100 ([final] is 0.5%)

200 mL 1X PBS (diluted from 10X stock with DPEC)

10ml Blocking Solution

.5 grams BSA fraction 5 (final 5%)

.05 grams Roche blocking solution (final .5%)

In PBS

  • PBST

1L 1X PBS (diluted from 10X stock with DPEC)

1ml Tween-20 ([final] is 0.1%) (notice this differs from J’s .01%)

  • PBT

10 g BSA Frac V

1 mL Tween 20

10 mL 100 mM EDTA

1 L 1X PBS (diluted from 10 X stock with DPEC)

Store 4 C

  • Labeled Tyramide Amplification Soln

Add 150 uL of high quality anhydrous DMSO (provided in kit) to 1 vial labeled tyramide supplied in kit

Divide into 10 uL aliquots, store -20 C dessicated.

  • Preparation/ Important Notes
  • Spray down work area with RNAse Zap
  • Rinse all glass histology containers with milli-q, then with RNAse Zap, and rinse again in milli-q before adding solvents.
  • Set hybridization oven precisely to hybridization temperature.
  • Warm Proteinase K buffer at 37 C
  • Purchased labeled probes should be aliquoted when received, since their efficiency decreases with multiple freeze and thaw cycles. FITC or DIG-labeled probes purchased from Exiqon usually have a concentration of 25 uM. Dilute the probe 1:10 with DPEC water to 2.5 uM final concentration. Add 2 uL of the diluted probe (5 pmoles) to 150 uL hybridization soln. The amount of probe used is critical for a successful ISH experiment.
  • The optimal hybridization temp is 20-25 C below the Tm of the probe. The Tm of each probe should be reported with the production information sheet for that probe. If not, call Exiqon.
  • All PBS washes are prepared with DPEC
  • Protocol
  1. Before Starting:
  2. Set oven to 60 degrees – check periodically to make sure correct temp, notice the display is not completely accurate
  3. Solutions:
  4. 4% PFA in PBS
  5. 3% H202 in PBS
  6. Hybridization buffer
  7. Acetylation solution
  8. Protease K solution
  9. .2% glycine solution
  10. .5% Triton solution
  11. 5% BSA in PBS
  12. PBT (day 2)
  13. PBST (day 2)
  14. RNase down all histology cups as described above. Deparaffinize slides 2x 10 min in Xylene.
  15. Begin rehydration through ethanol series
  16. 100% EtOH, 3-5 min
  17. 99% EtOH, 3-5 min
  1. 90% EtOH, 3 min
  2. 70% EtOH, 3 min*
  3. at this point in protocol, add proteinase K to prepared buffer. 200 mL buffer for a [final] of 5 ug/mL, place at 37 C (stock solution is 10ug/ul): add 100ul Proteinase K
  4. 50% EtOH, 3 min
  5. 25% EtOH, 3 min
  6. Remove 4% PFA from 4 C and equilibrate in fume hood
  7. Turn on hybridization oven and set to hybridization temp if not already done so. Place hybridization chamber (containing moist towels to maintain moisture) in oven to equilibrate.
  1. Place slides in DPEC H20, 1 min
  2. Place slides in 3% H2O2/PBS soln for 10 min in fume hood.
  3. Wash slides 3x 2 min in PBS.
  4. Place slides in Proteinase K soln for 5 min 37 C.
  5. Remove Proteinase K soln and add 0.2% Glycine soln for 1 min.
  6. Rinse in PBS 2 x 30s
  7. Place pre-hybridization soln in the hybridization oven (37C) to warm.
  8. Add ~1 mL of 4% PFA per section in fume hood for 10 min. Do not let PFA evaporate during this incubation time. After 10 min, remove PFA and let excess evaporate in fume hood.
  9. Wash 2x 5 min PBS, remove and add fresh PBS
  10. Begin pre-hybridization:
  11. Draw large square around all sections with wax pen: allow to dry before adding solution

Add ~500 uL of hybridization soln per slide. Put slides in a humidified chamber to avoid evaporation. Incubate at hybridization temperature (37C) for 90-120 min.

Before hybridization, dilute probes in hybridization soln to get a final concentration of 0.625 uM and heat probes to 60 C for 5 min to denature the probes.

  1. Begin hybridization: Remove excess pre-hybridization soln.
  2. Allow to dry, make small sections with wax pen
  3. Add 100 uL of probe soln (for lung/hearts) and 8 ul for vessels. Incubate overnight with probe at 37 C.
  4. The temperature of this incubation is dependent on the calculated Tm of the LNA probe (20-25 C below the calculated Tm).
  5. Place 0.2X SSC and PBS in the cold room overnight for the next day.
  6. Begin washing:
  7. Wash 1x 5 min in 0.2X SSC at 4C.
  8. Transfer to PBS
  9. Wash 1X PBS at 4C.
  10. The following steps begin the tyramide signal amplification kit directions (as determined by manufacturer).
  11. Make blocking solution supplied with tyramide amplification kit:
  12. 1% in PBS make only what is needed
  13. Block slides with 1% blocking solution for 30 min in cold room.
  14. Depending on timing, could alternatively try blocking 60-90 minutes and doing antibody over night.
  15. Incubate with anti-DIG-HRP antibody from Abcam diluted 1:50 in blocking solution at RT for 1hr.
  16. Prepare amplification buffer/ H2O2 stock soln by adding 1 uL H2O2 soln to 200 uL amplification buffer (both provided in kit). Now dilute this stock soln 1 to 100 to get working stock. (need to thaw amplification buffer)
  17. Quick rinse, followed by 3x 5 min washes in PBT at 4C, rocking.

From this point forward, everything is light sensitive, cover with foil!

  1. Prepare tyramide soln from kit using the amplifcation buffer working stock (2nd stock). Tyramide soln aliquots should be stored -20 C.

Use 1 uL tyramide soln per 100 uL amplification buffer 2nd stock. Incubate 12 min RT.

  1. Discard tyramide soln. Wash slides 3x 15 min in PBST at RT. Wash well! You can wash all day here if necessary!.
  2. Continue with 2x 10 min washes in PBST and 1x 10 min wash in PBS at RT.
  3. Begin mounting: Shake prolong gold with Dapi well before use. Add 2 drops of Prolong Gold and mount with coverslip. Let slides dry overnight at room temp. (can take a quick peak right away, but image will be better then next day) Visualize with fluorescence scope. Seal with nailpolish and store 4 C protected from light.

Changes:

No baking at 55 before starting protocol

Slightly longer pre-incubation and hybridization with probe

Block over night in cold room using invitrogen’s blocking solution and plastic cover slip

Increase acetylation time to 5 minutes.

Try higher probe concentration on the hearts.

Remove acetylation incubation and the subsequent washes in PBS.

Prehybridization at 37C rather than 55C.

Heat probes to 60C before the hybridization.

Hybridized overnight at 37C

Only wash once with 0.2X SSC at 4C (reduced stringency)

Block for 30 min.

Incubate with anti-DIG-HRP antibody for 1 hr.

Use a final concentration of 0.01% Tween 20 for PBST and PBT solutions.

Use new bottle of formamide.

Use 3 different probe concentrations (0.3125 uM, 0.625 uM, and 1.25 uM).

Incubate with anti-DIG-HRP antibody diluted 1:50 (used more antibody).