SAMPLE PREPARATION
Collect your sample and proceed to following steps, depending on the size of your sample. To know if your sample is considered small or large, here are some examples previously tested in the lab:
| Stage | ≤ GW6 | GW7-GW8 | GW8.5-GW9.5 | GW10-GW14 |
| Dissection | no | no | yes | yes |
| Fixation | O/N | 2 days | 4 days | 7 days |
| Size | Small | Large | Large | Large |
- For GW6 to GW8 embryos, fix in 4% paraformaldehyde (PFA). For older stages, acquisitions of whole embryo are not possible and therefore the structure of interest should be dissected After dissection re-fix in 4% PFA overnight at 4°C.
Store samples in PBS at 4°C until use.
DEHYDRATION/BLEACHING/REHYDRATION
If your sample is large or/and with blood hematoma proceed to this step, otherwise go directly to blocking:
- Dehydrate your sample in Methanol/PBS: 50% MeOH, 80% MeOH, 100% MeOH. 1h30 for each step with agitation and at room temperature (RT).
- Transfer to a solution of MeOH+6% H2O2 to bleach your sample. Incubate overnight at 4°C and protected from light.
- Rehydrate in: 100% MeOH x2, 80% MeOH (in PBS), 50% MeOH, PBS. 1h30 for each step with agitation and at RT.
Store samples in PBS at 4°C until use.
BLOCKING AND PERMEABILISATION:
- Incubate your sample in PBSGT for 24 hr to 4 days with rotation at RT. This incubation depends on the size of your sample: the larger, the longer.
(PBSG-T: 0.2% gelatin, 0.5% TritonX100 in PBS)
ANTIBODY INCUBATION:
All antibody incubation steps are performed at 37°C to increase antibody penetration. Depending on the size of your sample, your antibody, or the type of tissue you are using, you might need to adjust the incubation time or temperature. Here, we give incubation time for a small or a large samples.
- Incubate with primary antibodies in PBSGT + 0.1% saponin (10 µg/mL) for 7 days (small samples) or 2 weeks (large samples) at RT with rotation (70 rpm) and at 37°C.
- Rinse in PBS 1X +0.5% Triton (PBST), 6 times during 1 day at RT with rotation.
- Incubate with secondary antibodies in PBSGT+ 0.1% saponin (10 µg/mL) at 37°C 70 rpm overnight (small) or over 2 days (large). To avoid secondary antibodies precipitates in your solution, pass your final mix through 0.22 µm filter.
- Wash 6 times in PBST during 1 day at RT with rotation. Samples could be conserved at 4°C until clearing.
If your sample is too small to be hold in the ultramicroscope chamber, you can embed it in agarose (see TIPS section).
CLEARING: Transfer your sample in 15 ml TPP conical. This plastic is DBE resistant.
To clear our samples, we use the:
- 3DISCO protocol as followed:
- Incubate your samples in the following solutions at RT on a rotating wheel at 12 rpm: 50% THF overnight or 2 hr, 80% THF for 1-2 hr, 100% THF for 1-2 hr, 100% THF for 1-2 hr (again). Transfer in 100% Dichloromethane (DCM) during 30 minutes or until they have sunk. Transfer in 100% Benzyl ether (DBE) and wait for few hours for your sample to get transparent.
All Tetrahydrofuran (THF) solutions are diluted in H20. All these products need to be used under a hood and with protections.
Keep your sample in DBE at RT, protected from light in a glass bottle, NOT at 4°C.
- Methanol clearing protocol as followed: (adapted from iDISCO+ protocol, Renier et al. 2016)
- Incubate your samples in the following solutions at RT on a rotating wheel at 12 rpm: 20% MeOH, 40% MeOH, 60% MeOH, 80% MethOH, 100% MeOH, 100% MeOH; 1h each.
- 3 hr or Overnight incubation in 2/3 DCM+ 1/3 Methanol.
- Incubate in 100% DCM 30 minutes to wash out the MeOH.
- Transfer in 100% Benzyl ether (DBE) and wait for few hours for your sample to get transparent.
All Methanol (MeOH) solutions are diluted in PBS. All these products need to be used under a hood and with protections.
Keep your sample in DBE at RT, protected from light in a glass bottle, NOT at 4°C.
REAGENTS:
4% PFA : Paraformaldehyde (VWR- 1.04005.1000) in phosphate buffer 0.12 M (pH 7.2-7.4)
PBSGT-0.5X : 1X PBS, 0.2% Gelatin, 0.5%Triton X100 (SIGMA- X100)
Saponin (SIGMA-S4521)
Tetrahydrofuran Anhydre >99.9% (SIGMA -186562)
Dichloromethane (SIGMA-270997)
Benzyl ether (SIGMA-108014)
Methanol (VWR – 20847.360)
H2O2 30% (SIGMA- art.216763)
Agarose (ROTH- 2267.4)
10X TAE (Life Technologies- 15558026)
Gelatin (VWR-24350.262)
Thimerosal (SIGMA-T8784-5g)
Triton X100 (SIGMA- X100-500ml)
10X PBS (Life Technologies- 14200-083)
Di-sodium hydrogenophosphate (VWR- PROLABO 28028.298)
Sodium dihydrogenophosphate (VWR- PROLABO 28015.294)
SYLGARD® 184 SILICONE ELASTOMER KIT (DOW CORNING)
Hydroxyde de sodium pellets EMSURE® (VWR-1.06498.1000)
DENT Dentalon® plus powder en 100 g (PHYMEP- 13301006 05)
DENT Dentalon® plus liquid en 45 ml (PHYMEP-1330 0502 05)
MATERIAL
Incu-Shaker Mini (Benchmark)
Rotator SB3 (STUART)
Glass bottle for small samples: Art E160.1+ screw caps (E162.1)+PTFE septa (E163.1). All purchased from ROTH
Glass bottle for big sample: XC44.1+ screw caps (XC38.1): purchase from ROTH
Benchtype Fume Adsorber TAZ 19.000 (MEDITE) + carbon (Art. 46-6040-00)
0,22 μm Syringe Filter Rotilabo®, CME porosity, sterile Ø ext. 33 mm (ROTH- KH54.1)
Butyle gloves SHOWA 874R (ROTH)
StarGuard® PROTECT+ gloves (Starlab)
TIPS
If you want to know the protocol for our samples related to the tiff folder in the 3D database, please contact us. We will send you all the details of processing step.
You can perform your immunostaining in toto in 2 ml Safe-lock Eppendorf or 12/24 wells plate to adapt the volumes of antibody solution to the size of your sample. This can save you a lot of antibodies.
Always cover your plates and tubes with parafilm to avoid evaporation during the antibody incubation step.
If your sample is too small, the best is to embed your sample in 1.5% agarose in 1X TAE. To do so, melt your agarose in a microwave. Pour some agarose in a weighing tray and gel at 4°C. Then, position your sample on the tray and add (not too hot) melted agarose. Let it gel at 4°C and cut an agarose cube containing your sample with a razor blade and proceed to the clearing.
TROUBLESHOOTING:
The time of fixation in 4% PFA is very important: if it is too short, the immunostaining will be impaired. If it is too long you will not be able to clear your sample.
The incubation time with primary antibodies and the temperature of incubation can be adjusted. The general rule is that the longer the better. Some antibodies work only at 4°C or at RT. If your antibody works on sections, it should work on whole mount immunostaining.
Red (Cy3 ) and far Red (Cy5 or equivalent)-coupled secondary antibodies give nicer images than alexa488/FITC.
PREPARATION OF HISTOLOGICAL SOLUTIONS:
8% PFA (1 L)
Boil 1 L of distilled water under the chemical hood and add 80 g of paraformaldehyde.
Add 2 drops of NaOH 1 N and stir the solution.
Chilled on ice and filter through a pleated filter. Aliquot and keep at -20°C. The best is to prepare freshly this solution.
0.24 M Phosphate buffer, pH 7,2 (5 L)
Dissolve 36 g of NaH2PO4, 2H2O (sodium dihydrogenophosphate) and 344 g Na2HPO4, 12 H2O (di-sodium hydrogenophosphate) and complete the volume until 5 L with distilled water.
1 N NaOH (1 L)
Dissolve 40 g of sodium hydroxide in 1 L of water.
PBSG-T: 0.2% gelatin, 0.5% Triton in PBS, (1 L)
Dissolve by gentle heating 2 g of gelatin (VWR 24350.262) in 900 mL of distilled water.
Filter immediately (pleated filter) and wait for the solution to chill before adding 100 mL of 10XPBS, 20 mL of 25 % Triton X-100 and 0.1 g of Thimerosal.
25% Triton X-100 (500 mL)
Dissolve 125 ml of Trion X-100 in 375 ml of sterile distilled water with stirring.
Tips: Add TritonX-100 gently little by little. You can aliquot and keep it at 4°C.
10 mg/mL Saponin
Dissolve saponin in water at a final concentration of 10 mg/mL.