Wholemount Fluorescence ISH Protocol
(Adapted from Rahul Parnaik-Ragsdale Lab by Gina Elsen and Gokhan Dalgin-Prince Lab University of Chicago)
Embryos Fixation and Dehydration:
- Fix overnight at 4°C in 4% PFA.
- Rinse 2X in PBT for 5 minutes
- Dehydrate embryos through a series of methanol solutions at RT
- 30% MeOH in PBT for 5 minutes
- 60% MeOH in PBT for 5 minutes
- 100% MeOH in PBT for 5 minutes; replace with fresh 100% MeOH
- Incubate dehydrated embryos at --20°C overnight (at this point may be stored up to 1year).
DAY 1: Rehydration, ProK, Hybridization
- Rehydrate embryos through a series of methanol solutions at room temperature in 1.5ml tubes (optional: we usually transfer embryos in 48-well plate at this point till the end of the protocol)
- 60% MeOH in PBT for 5 minutes
- 30% MeOH in PBT for 5 minutes
- PBT for 5 minutes, 2X
- Wash once with 1XPBS 5 minutes
- Incubated embryos on rocker in 0.6% hydrogen peroxide in PBS for 10 minutes at RT (you can increase H2O2 amount upto 2% but the embryos become too soft) This step both bleaches the embryos and inactivates any residual peroxidase activity.
- Wash 3X in PBT, 5 minutes each.
4. Digest with 1X (10μg/ml) Proteinase-K at RT (ProK is at 100X (1mg/ml) keep stock in -200C). For 1X, dilute 100μl into 10ml PBT
No somites no digetion
1-6 somites 3 minutes
7-12 somites 5 minutes
12-18 somites 10 minutes
18 somites -24hpf 13 minutes
24hpf-36hpf 15 minutes
48hpf 45 minutes
72hpf 1hour and 15 minutes
- Post-fix embryos on rocker in 4% PFA for 1.5 hour at RT,
- Wash 4X in PBT, 5 minutes each, at RT.
- Remove PBT and replace with prehybridization solution (pre-HYB)
To Make Hybridization buffer (HYB-65%): In a 50 ml tube: Final Concentration
Formamide (stored at -200C) 32.5 ml.............................65%
20 x SSC/DEPC 12.5 ml.............................5x
Heparin @ 50 mg/ml (40C) Sigma H-3393 50μl.......................…….50μg/ml
20% Tween 250 μl...............................0.1%
0.5M Citric acid 920 μl...........................…to pH 6
DEPC/water to 50 ml (store in --20°C)
- Incubate embryos in 300ml pre-HYB in 70°C incubator for 1-3 hours.
- Remove pre-HYB and add appropriate volume of the riboprobe(s) to the HYB-tRNA (*Add 5mg tRNA to 10ml Hyb mix-tRNA Roche 10109495001). Incubate in a 70°C incubator overnight (minimum 12 hours).*
DAY 2: Post-hybridization Washes to Antibody Incubation:
(Note: Pre-warm solutions A, B, C, 2X SSC, 0.05X SSC at 700C).
- Remove probes (save the probes)
- Washes, in 700C incubator:
Wash A: 75% HYB + 25% 2X SSC, 10 minutes
Wash B: 50% HYB + 50% 2X SSC, 10 minutes
Wash B: 25% HYB + 75% 2X SSC, 10 minutes
2X SSC, 10 minutes
0.05X SSC, 30 minutes 2X
Note: Start thawing Western Blocking reagent (WBR) (#1921673, Roche) (Consider this as 10X stock and store in aliquots at -200C)
- Washes at RT on a shaker:
Wash D: 75% 0.5X SSC + 25% PBT, 10 minutes
Wash E: 50% 0.5X SSC + 50% PBT, 10 minutes
Wash F: 25% 0.5X SSC + 75% PBT, 10 minutes
3X PBT, 5 minutes
- Blocking step: incubate embryos in 1X WBR (dilute from the 10XWBR in PBT and filter it!!! Fisherbrand .22μm Cat# 09-719A) for 2 hours at RT.
5. Add anti-Dig-POD *(The reagent is an anti-digoxigenin antibody from sheep, Fab fragments, conjugated with polymerized horse-radish peroxidase [POD(p)]. (Roche* 11 633 716 001*)* final concentration of 1:100-1000 in filtered 1X WBR. Incubate on rocker overnight at 4°C.
Note: Test appropriate antibody concentration so far 1:100-1000 range works for us.
DAY 3: Post-10Antibody Washes :
1. Remove the anti-Dig-POD (HRP) 10 Antibody
2. Rinse 2X in PBT, and wash 6X in PBT, 10 minutes each
- Wash in PBT overnight at 4°C on rocker OR
- Proceed to tyramide reaction. If time is not limiting, it is better to do the overnight wash to produce the cleanest possible results.
DAY 4: Tyramide reaction:
1. Preincubate in AB (amplification buffer) for 10 minutes with rocking.
- Use TSA Plus Cyanine3 System (Perkin Elmer NEL74400 1KT) to detect Dig-probes.
- Make TWS (tyramide working solution) just prior to the signal detection. To make TWS dilute the tyramide stock 1:100-1000 in 0.0015% H2O2/ amplification buffer (AB). Need about 200μl of TWS /well. Do serial dilution to make 0.0015% H2O2/AB: first make 0.15% H2O2 in AB (1:200 from 30% stock H2O2), then make AB/0.0015% H2O2 (1:100 from the 0.15% stock H2O2).
2. Incubate embryos on rocker in RT for 1 hour in Cy3 Plus tyramide, and protect from light.
3. Wash for 15 minutes in PBT, 3X in RT.
4. Can look at embryos on an epifluorescence or confocal microscope at this point or continue with detection of next hapten. Wash longer if high background.
Second color reaction:
Antibody labeling for the 2nd probe:
1. Incubate 10 minutes in 0.1 Glycine, pH2.2 (low pH in general cause the initial 10 antibody to be stripped away) (Notes: This step is optional)
2. Wash 5X in PBT
3. Block 2hours at RT in 1X WBR (filtered!!!)
4. Incubate in anti-Fl-HRP 10 Antibody (Perkin Elmer NEF 710) (1:100-1000 in 1X WBR) overnight at 40C.
DAY 5: 2nd TSA reaction
1. Remove 10 Antibody (for the 2nd color reaction)
2. Rinse 2X in PBT, and wash 6X in PBT, 10 minutes each (Notes: Wash longer or overnight if possible)
3. Incubate in Amplification Buffer (we use home brew amplification buffer) for 15 minutes at RT.
(Notes: Make Tyramide Working Solution (TWS) just prior to the signal detection. To make TWS dilute the tyramide stock 1:100-1000 in 0.0015% H2O2/ amplification buffer (AB). Need about 200μl of TWS /well. Do serial dilution to make 0.0015% H2O2/AB: first make 0.15% H2O2 in AB (1:200 from 30% stock H2O2), then make AB/0.0015% H2O2 (1:100 from the 0.15% stock H2O2).
- Incubate for 1 hour in FITC-TSA (Perkin Elmer). (We use home-made Fluorescein-tyramide diluted in home brew amplification).
- Wash 3 X for 15 minutes (or longer and observe color reaction).
Solutions and Reagents
Home Brew: For 1 L
To 1 L of DEPC-treated PBS add
0.1 M Imidazole 6.8g Imidazole
Adapted from Vize et. al., NATURE PROTOCOLS
VOL.4 NO.6 2009 p975 and Rahul Parnaik-Ragsdale Lab by Gokhan Dalgin
Basic idea: react reactive succinimidyl ester with tyramine hydrochloride under
anhydrous conditions and with correct stoichiometry.
So, use fresh (anhydrous) DMF.
1) Make FL-ester solution at 10mg/ml in DMF:
100mg NHS-fluorescein (MW=473, NHS-Fl is moisture-sensitive equilibrate vial to RT before opening read manufacturers product information!!!) added to 10ml DMF
2) Make DMF-TEA solution:
3) Make reactive tyramine solution:
50mg tyramine hydrochloride added to 5ml TEA-DMF solution.
4) Mix reagents:
10ml FL-ester solution
3.425ml tyramine solution
React for 2hr at room temp in the dark (ie, cover with foil or place in a drawer)
Then add 11.5 ml 100% ethanol. This makes a 100x stock.
Make 1ml aliquots. It is good for about a year in -20 or -80
Just to point out the cost diff between various dyes:
100mg Fluorescein Ester = $110
1mg Cy3/Cy5 ester = $214
DMF Anhydrous, Acros CAS: 68-12-2, Product Code 610941000
Triethylamine 99% prue, Acros CAS:121-44-8, Product Code 157911000
Tyramine hydrochloride 99% Sigma CAS: 60-19-5 Product Number T2879 1G
Ethanol 200 proof Sigma CAS: 64-17-5 Product Number E7023
NHS-Fluorescein, 100mg Thermo Scientific Product number 46410
Cy3 Mono NHS Ester GE healthcare/Amersham PA13101