Whole-mount in situ hybridization protocol for Platynereis larvae for 3D microscopy

Group Balavoine/Vervoort (16/1/14), Institut Jacques Monod, Paris, adapted from Jékely Lab

In situ Hybridization Day 1

PRETREATMENT

1. Transfer embryos to small mesh filter cups in a tip box lid with 50 ml 75% MeOH/25% PTw.
   Use 200-300 larvae per probe for each stage.
   Shake at 300 rpm until the Proteinase K step.
2. On shaker in fumer hood, rehydrate larvae through:
   50 ml 75% MeOH/25% PTw 5 min
   50 ml 50% MeOH/50% PTw 5 min
   50 ml 25% MeOH/75% PTw 5 min
   50 ml 100%PTw 5 min
   50 ml 100%PTw 5 min
3. Prepare 3 solutions in tip box lids:
   (1) Proteinase K: 250 μl 20mg/ml ProK in 50 ml PTw.
   (2) Glycine 2mg/ml.    Add to 50 mL PTw.
   (3) 4% paraformaldehyde in PTW 40 mL: 12,5 ml 16% PFA in 40 mL PTw.
4. Digest larvae with Proteinase-K without shaking:

5. Wash larvae briefly (<1min) 2mg/ml glycine in PTw (make fresh every time).

6. Try the following steps for in situ with embryonic stages (pre-hatching) otherwise go straight to Step 7:
   1. Wash with 1% triethanolamine in PTw until embryos settle.
   2. Add 3 uL/ml acetic anhydride to an aliquot of 1% triethanolamine in PTw and vortex thoroughly.
      Add to embryos immediately.
   3. To the same aliquot of 1% triethanolamine/PTw/Acetic anhydride add an additional 3 ul/ml and vortex thoroughly.
      Add to embryos immediately.
   4. Wash 2x in PTw

7. Post-fix larvae in 4% PFA in PTw at room temperature for 20 minutes, shaking at 300rpm.
8. Wash 5x 5min in 100% PTw 30 mL, shaking at 300rpm.
9. Transfer larvae from nets back to 2 ml eppies using dissecting microscope.
   Carefully wash all larvae down from the sides of the mesh basket.

HYBRIDIZATION

10. Step larvae into Hyb.
    Replace PTw supernatant with 2ml PTw 66% / Hyb 33%
    mix by inverting.
    Allow larvae to settle.
    Repeat with PTw 33% / Hyb 66% then Hyb 100%.
    Split larvae into different tubes for different probes, combine different stages of larvae etc. Use the small mesh baskets in 2ml eppies.
11. Incubate larvae in 100% Hyb. mix in 65°C incubating oven for 1 – 4h.
12. Prepare DIG-labeled RNA probes in 200 μl Hyb mix in flat bottom tubes (total final volume), denature at 85°C for 10 minutes.
    (For most genes, between 200 ng probe in 200 μl of Hyb is good; this should be optimized on a probe-by-probe basis; concentration of probes for fluorescent in situs are higher.).
13. Transfer both probe tubes and larvae tubes from 65°C oven to 65°C bain-Marie.
    Transfer mesh baskets to probe tubes taking care of efficient draining each time.
    Return larvae to 65°C oven, with agitation
    It is very important that larvae stay at 65°C from now until the end of the Day 2 formamide washes to ensure that the probe hybridizes specifically.
14. Hybridize larvae at 65°C overnight (Aim for ~16h or more).

In situ Hybridization Day 2

1. Make formamide-containing wash solutions & pre-warm all wash solutions (4X, 2X and 0.2X, recipes below) to 65°C in water bath.
   During formamide washes maintain samples at the appropriate hybridization temperature – eg. transfer quickly from oven to water bath; transfer quickly mesh baskets from one tube to another.
2. Remove probe/Hyb Buffer solution from samples (store probes for re-use of desired).
3. Wash with 800 μl of 4X wash solution at hybridization temperature for 15 min.
4. Repeat 4X wash.
5. Repeat 4X wash.
6. Wash with 1ml of 2X wash solution at hybridization temperature for 15 min.
7. Repeat 2X wash.
8. Wash with 1 ml of 0.2X wash solution at hybridization temperature for 15 min.
9. Repeat 0.2X wash.
10. Repeat 0.2X wash.
11. Exchange for 800 μl of room temperature 0.2X SSC with 0.1% Tween (NO FORMAMIDE).
    Place tubes at room temperature (you can now transfer the the sample back to 1.5 ml eppies without filters, as the larvae will sink again).
    Rotate larvae on a Nutator for 20 min
    The salt (SSC) concentration can be reduced further if there is a problem with background.
12. Repeat wash for 20 min.
13. Repeat wash for 20 min.
14. Wash 5 min with PTw
15. Incubate in blocking solution (PTw + 5% sheep serum) on Nutator at room temp for two hours.
16. Replace blocking solution with 300 μl PTw with 1/5000 anti DIG antibody, 1:500 anti-acetylated tubulin (mouse).
17. Incubate larvae at 4°C overnight on Nutator in cold room.

In situ Hybridization Day 3

1. Wash samples with 1ml PTw at room temperature 8X 20min (or minimum 2 hrs)
2. Wash 2x 5 minutes in AP buffer without MgCl2
3. Wash 1x 10 minutes in AP buffer WITH MgCl2
4. Prepare staining buffer (6% PVA in 1X AP Buffer with 50mM MgCl2; 2.25 μl of 100 mg/ml NBT and 3.5 μl of 50 mg/ml BCIP per ml of 1X AP)
5. Transfer embryos to staining buffer in 6-well plate, 2 ml staining buffer per well.
   Keep in the dark at 4°C while developing colour.
   Check for staining regularly (eg after 30 min, 1 h, 2 h, 4 h etc).
6. Stop colour development by 1X 5 min wash in STOP buffer.    Larvae can be stored here at 4°C if you need.
7. Wash samples 3X 5min in PTw.
   Store at 4°C, or continue on to secondary antibody addition.
8. Incubate larvae in 250 μl PTw with 5% sheep serum and secondary antibody (TRITC anti mouse, 1:250) plus DAPI 1ug/ml overnight at 4°C on Nutator
   (wrap tubes in alfoil to keep them in the dark).

In situ Hybridization Day 4

(Try not to expose samples to too much light)

1. Wash samples in 1ml PTw 3x 10 min at RT
2. Wash samples in 1ml PTw 1x 30 min – 1h at RT on Nutator (wrap in foil).
   Larvae can be stored in PTw at 4°C until you are ready to transfer to 97% TDE.
3. Transfer into TDE:
   1. Prepare 33% TDE/67% PTw and 66% TDE/34% PTw, vortex to mix.
      Prepare a 24-well plate: From left to right; 1 column with net from in situ robot, 1 column with 700ul 66% TDE/34% PTw, 1 column with 700ul 97% TDE, 1 column with 1ml 97% TDE.
   2. Add larvae in 600ul PTw to net in 1st column.
      Suck PTw off from under the net using a P1000 and replace with 700ul 33% TDE/67% PTw.    Incubate on Nutator 10min at room temp.
      Transfer larvae in net to 66% TDE/34% PTw well using forceps, incubate on Nutator 10min at room temp.
      Transfer larvae in net to 97% TDE, incubate on Nutator 10min at room temp.
      Transfer larvae in net to 1ml 97% TDE, incubate on Nutator 10 min at room temp.
   3. Remove larvae in 97% TDE from net under dissecting scope and transfer to a fresh 24-well plate for storage at 4°C in dark in 97% TDE, label plate well (eg Name, date, genes, primary/secondary antibodies, larval stages).

Product Ordering Information

In situ solutions

* Solutions should be autoclaved to sterilize
+ Solutions should be made with RNase/DNase-free water

*10xPBS

18.6 mM NaH2PO4.H20 (2.56g/Liter)

84.1 mM Na2HPO4.2H2O (14.97g/Liter)

1,750 mM NaCl (102.2g/Liter)

Mix phosphates in 800mL dH20.
Check to ensure pH is near 7.4 (within .4).
If significantly different, start over.
Adjust pH to 7.4 with HCl or NaOH.
Add NaCl and remaining water.
Make sure that you are dealing with the correct phosphate stock powders (monohydrate vs. dihydrate etc).
The masses will change depending on this.

*PTw

100mL 10x PBS stock

895mL dH20

5mL 20% Tween-20

_Dilute 10x PBS to 1x.
DEPC treat, autoclave and cool.
Add tween.

+ Heparin

Make a stock of 50 mg/ml in H2O, store at -20°C

+ Hybridization Mix

50% formamide (Fluka, ultra pure)
5X SSC
50 µg/ml heparin
0.1%Tween20
5 mg/ml torula RNA

store at -20°C.

For 50 ml of Hyb. Mix:

stock Hyb-mix
Formamide 100 % 25 ml
SSC 20 x 12.5 ml
Heparin 50 mg/m l50 µl
Torula-RNA (Sigma) solid 250 mg
Tween20 10 % 500 µl
H2O ad 50 ml

*20x SSC pH 7.0

175.3g NaCl
88.2g NaCitrate
1L dH20

Mix salts and dH20.
pH to 7.0, DEPC treat and autoclave.

*20x SSC pH 4.5

175.3g NaCl
88.2g NaCitrate
1L dH20

Mix salts and dH20.
pH to 4.5, DEPC treat and autoclave.

+Formamide Wash Solutions

4X SSC wash (50ml)
Formamide 25ml
20X SSC pH 7 10ml
20% Tween-10    250µl
H2O 14.75ml

2X SSC wash (50ml)

Formamide 25ml
20X SSC pH 7 5ml
20% Tween-10    250µl
H2O 19.75ml

0.2X SSC wash (50ml)

Formamide 25ml
20X SSC pH 7 500µl
20% Tween-10    250µl
H2O 24.25ml

*1 M NaCl (500mL)

29.22g NaCl
500mL dH20

Autoclave to sterilize.

*1M MgCl2 (250mL)

50.75g MgCl2 (if hexahydrate only)
250mL dH20

Mix and autoclave to sterilize

*1M Tris pH 9.5 (500mL)

60.57g Tris
500mL dH20

Mix and autoclave to sterilize

+5X AP Buffer (200ml)

1M Tris pH 9.5 100ml
5M NaCl 20ml
H2O 80ml

Store at room temperature.

+1X AP Buffer (50ml)

5X AP Buffer 10ml
H2O 39.75ml
20% Tween-20 250µl

+1X AP Buffer with MgCl2 (50ml)

5X AP Buffer 10ml
H2O 37.25ml
1M MgCl2 2.5ml
20% Tween-20 250µl

+Staining Buffer (Eg for 10 tubes)

Final concentrations – MgCl2 50mM
100mg/ml NBT à 4.5µl/ml
50mg/ml BCIP à 3.5µl/ml
pH 9.5!!

1. Make 12% polyvinylalcohol (PVA) in 1X AP Buffer with MgCl2.
   Heat in short bursts in microwave to dissolve, watch carefully, this solution boils over very fast!! Make 20ml at a time.
2. Make half the amount of 1X AP Buffer you will need (eg 5mL).
3. To the 1X AP Buffer, add 4.5ul/ml NBT and 7ul/ml BCIP.    (Eg for 5ml, add 22.5 ul NBT, 35ul BCIP).This solution is light sensitive.
4. Filter NBT/BCIP solution through 0.22µM syringe filter.
5. Combine 5ml 12% PVA with 5mL filtered NBT/BCIP solution (1:1), mix gently but thoroughly.
   Check pH is close to 9.5 (pH is important!).
6. Aliquot this staining solution into 1ml/24 well plate well, then add the larvae from the eppies in minimal volume (50-100µl) of 1X AP Buffer with MgCl2.
   Gently mix larvae in plate (on orbital shaker table is nice).
   Alternatively, remove as much 1X AP Buffer with MgCl2 from samples in eppies and add 1ml staining buffer, then transfer 1ml samples in staining buffer to well-plate.

+STOP Buffer (50ml)

2M Tris pH 7.5 2.5ml
5M NaCl 1ml
1M MgCl2 2.5ml
20% Tween20 250µl
H2O to 50mL.

Store solution at 4C.