In situ Protocol (final) ( Click to Download )

J. Rudnick 11/08

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

U6 99002-01 $225 Tm: 75
Scramble 99004-01 $225 Tm: 78
Hsa-mir-24 18121-01 $375 Tm: 80
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 25mg Roche 03115836001 $42
Prolong Gold with Dapi Invitrogen P36931 $128

Hybridization temp: J: 22-25 below calculated tm: for this set, use 55 degrees
Probes come at 25µM concentration
Dilute 5µl probe + 45µl RNAse free water to give 2.5µM. 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

•500 mL of 5 M NaCl
146.1 g NaCl
500 mL DPEC

•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

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 37oC 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 et al for alternative)
5 mL formamide ([final] is 50%)
2.5 mL 20X SSC ([final] is 5X)
120 µL 25 mg/mL yeast tRNA ([final] is 300 µg/mL)
200 µL of 50X Denhardt’s Soln ([final] is 1X)
50 µL 20% Tween-20 ([final] is 0.1%)
2.13 mL DPEC H20
Divide into 150 µl aliquots and store -20oC.
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 µL 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)


•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%)

1L 1X PBS (diluted from 10X stock with DPEC)
1ml Tween-20 ([final] is 0.1%) (notice this differs from J’s .01%)

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 4oC

•Labeled Tyramide Amplification Soln
Add 150 µL of high quality anhydrous DMSO (provided in kit) to 1 vial labeled tyramide supplied in kit
Divide into 10 µL aliquots, store -20oC 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 37oC
•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 µM. Dilute the probe 1:10 with DPEC water to 2.5 µM final concentration. Add 2 µL of the diluted probe (5 pmoles) to 150 µL hybridization soln. The amount of probe used is critical for a successful ISH experiment.
•The optimal hybridization temp is 20-25oC 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:
        1. Set oven to 60 degrees – check periodically to make sure correct temp, notice the display is not completely accurate
        1. Solutions:
            1. 4% PFA in PBS
            1. 3% H202 in PBS
            1. Hybridization buffer
            1. Acetylation solution
            1. Protease K solution
            1. .2% glycine solution
            1. .5% Triton solution
            1. 5% BSA in PBS
            1. PBT (day 2)
            1. PBST (day 2)
    1. RNase down all histology cups as described above. Deparaffinize slides 2x 10 min in Xylene.
    1. Begin rehydration through ethanol series
        1. 100% EtOH, 3-5 min
        1. 99% EtOH, 3-5 min
        1. 90% EtOH, 3 min
        1. 70% EtOH, 3 min*
            1. at this point in protocol, add proteinase K to prepared buffer. 200 mL buffer for a [final] of 5 µg/mL, place at 37oC (stock solution is 10µg/µl): add 100µl Proteinase K
        1. 50% EtOH, 3 min
        1. 25% EtOH, 3 min
            1. Remove 4% PFA from 4oC and equilibrate in fume hood
            1. 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
    1. Place slides in 3% H2O2/PBS soln for 10 min in fume hood.
    1. Wash slides 3x 2 min in PBS.
    1. Place slides in Proteinase K soln for 5 min 37oC.
    1. Remove Proteinase K soln and add 0.2% Glycine soln for 1 min.
    1. Rinse in PBS 2 x 30s
        1. Place pre-hybridization soln in the hybridization oven (37oC) to warm.
    1. 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.
    1. Wash 2x 5 min PBS, remove and add fresh PBS
    1. Begin pre-hybridization:
        1. 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 (37oC) for 90-120 min.
          Before hybridization, dilute probes in hybridization soln to get a final concentration of 0.625 µM and heat probes to 60oC for 5 min to denature the probes.
    1. Begin hybridization: Remove excess pre-hybridization soln.
        1. Allow to dry, make small sections with wax pen
        1. Add 100 µL of probe soln (for lung/hearts) and 8 µl for vessels. Incubate overnight with probe at 37oC.
        1. The temperature of this incubation is dependent on the calculated Tm of the LNA probe (20-25oC below the calculated Tm).
        1. Place 0.2X SSC and PBS in the cold room overnight for the next day.
    1. Begin washing:
    1. Wash 1x 5 min in 0.2X SSC at 4oC.
    1. Transfer to PBS
    1. Wash 1X PBS at 4oC.
        1. The following steps begin the tyramide signal amplification kit directions (as determined by manufacturer).
        1. Make blocking solution supplied with tyramide amplification kit:
            1. 1% in PBS make only what is needed
    1. Block slides with 1% blocking solution for 30 min in cold room.
        1. Depending on timing, could alternatively try blocking 60-90 minutes and doing antibody over night.
    1. Incubate with anti-DIG-HRP antibody from Abcam diluted 1:50 in blocking solution at RT for 1hr.
        1. Prepare amplification buffer/ H2O2 stock soln by adding 1 µL H2O2 soln to 200 µL amplification buffer (both provided in kit). Now dilute this stock soln 1 to 100 to get working stock. (need to thaw amplification buffer)
    1. Quick rinse, followed by 3x 5 min washes in PBT at 4oC, 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 -20oC.
      Use 1 µL tyramide soln per 100 µL 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!.
    1. Continue with 2x 10 min washes in PBST and 1x 10 min wash in PBS at RT.
    1. 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 4oC protected from light.

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 37oC rather than 55oC.
Heat probes to 60oC before the hybridization.
Hybridized overnight at 37oC
Only wash once with 0.2X SSC at 4oC (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 µM, 0.625 µM, and 1.25 µM).
Incubate with anti-DIG-HRP antibody diluted 1:50 (used more antibody).