• FlyNap® Anesthetic (Carolina®, 173025).
• Vaseline (Panreac, cat. no. 151757).
• NaCl (J.T.Baker, cat. no. RS485500036).
• MgCl2 (Panreac, cat. no. 131396.1210).
• KCl (J.T.Baker, cat. no. C43340).
• CaCl2 dehydrate (Panreac, cat. no. 129903731).
• NaH2PO4 (Panreac, cat. no. 121677.1211).
• HEPES (Roche, cat. no. 10172944103).
• Sucrose (J.T.Baker, cat. no. 1400801832).
• D-(+)-Trehalose dehydrate (Sigma, cat. no. SLBJ7735V).
• NaHCO3 (Panreac, cat. no. 131638).
• Paraformaldehyde (methanol-free; Sigma-Aldrich cat. no. P6148).
CAUTION. Paraformaldehyde is flammable and very toxic.
• 10X PBS, pH 7.0 (Roche, cat. no. 11666789001).
• 70% ethanol.
• Absolute ethanol (Panreac, cat. no. 131086.1214).
• Triton X-100 (Roche, cat. no. 10789704001).
CAUTION. Triton X-100 is harmful.
• Bovine Serum Albumin (BSA) Fraction V (Roche, cat. no. 10735078001).
• Donkey serum (Sigma Aldrich, cat. no. D9663).
• Primary antibodies: sheep anti-Muscleblind27 and rabbit anti-GFP (Invitrogen, cat. no. G10362).
• Secondary antibodies: Biotin-conjugated anti-sheep (Thermoscientific, cat. no. 31840), and anti-rabbit FITC (Sigma-Aldrich, cat. no. F9887).
• Streptavidin–Texas-red (VECTOR, cat. no. SA5006).
• Elite ABC-Peroxidase Staining Kit (standard; Vectastain, cat. no. PK-6100).
• Phalloidin (Sigma-Aldrich, cat. no. P1951).
• DM1 DNA oligo probe (5'-/5Cy3/CAG CAG CAG CAG CAG CAG CA/3Cy3Sp/-3') (Integrated DNA technologies).
• Formamide (Sigma-Aldrich, cat. no. 47671)
CAUTION. Formamide is toxic and flammable.
• AG® 501-X8 (D) Resin (Bio-RAD, cat. no. 142-6425).
• Tris base (cat no. Promega, cat. no. H5135).
• EDTA (Panreac, cat. no. 131669.1211).
• Dextran sulfate (Sigma, cat. no. D8906-10G).
• 50X Denhart´s solution (Sigma, cat. no. D9905-5ML).
• 10 mg/ml Herring Sperm DNA (Promega, cat. no. D1815).
• Vectashield® antifade mounting medium (VECTOR, cat. no. H-1200).
• miRNeasy® Mini Kit (Qiagen, cat. no. 1086311).
• Chloroform (JT Baker, cat. no. UN1888).
• 10X DNase Reaction Buffer (Roche, cat. no. EVH208).
• DNase I Recombinant (Roche, cat. no. EVH208).
• dNTPs [10 mM] (Thermo Scientific cat. no. R0192).
• 10X Hexamers (Roche, cat. no. 26590420).
• 5X First Strand Buffer (Invitrogen, cat. no. 1907545).
• 0.1 M DTT (Invitrogen, cat. no. 1907545).
• RNaseOUTTM Recombinant (Invitrogen, cat. no. 1900300).
• SuperScriptTM II (Invitrogen, cat. no. 1907545).
Throughout the reagent setup use RNase-free MilliQ water
• Artificial hemolymph solution.
Prepare the following solutions:
- 1.08 M NaCl, 0.08 M MgCl2, 0.05 M KCl, 0.02 M CaCl2 dehydrate, 0.01M NaH2PO4 and 0.05 M HEPES (solution A).
- 0.1 M Sucrose (solution B).
- 0.1 M D- (+)- Trehalose dehydrate (solution C).
- 0.25 M NaHCO3 (solution D).
Prepare artificial hemolymph (AH) solution as follows:
Mix 10 ml of solution A, 10 ml of solution B, 5 ml of solution C and 1.6 ml of solution D. Add autoclaved MilliQ water until 100 ml of total volume. Oxygenate the solution for 15-20 min by air-bubbling prior to the dissection and set the pH at 7.1. Use this solution within few hours.
CRITICAL. Store all the solutions at 4°C. Bring to RT before use. Change the 1X oxygenated artificial hemolymph after few hours because heart beating depends on proper oxygen supply.
• Paraformaldehyde 4% (PFA 4%)
Use 1X PBS to dissolve the paraformaldehyde powder. To dissolve the PFA in the PBS, use heat and a few drops of 10 N NaOH. The pH of the PFA should be 7.3-7.4. Aliquoted PFA can be stored at -20°C for several months. Dispose the PFA appropriately.
CAUTION. PFA is very toxic and flammable.
CRITICAL. Use fume hood. Do not heat higher than 60°C to dissolve the PFA. Do not add more than 2 drops of 10 N NaOH to dissolve it. Cover the solution to avoid evaporation.
• Washing solution.
Add Triton X-100 to 1X PBS to have a final concentration of 0.3%. Washing solution can be stored for some days at 4°C.
CAUTION. Triton X-100 is toxic.
• Tris-HCl buffer (pH 8.0)
For a total volume of 1 L, dissolve 121.4 g of Tris base in water. Adjust the appropriate pH with 37% HCl. The solution can be stored at room temperature (RT).
• 0.5 M EDTA (pH 8.0)
For a total volume of 1 L, dissolve 186.1 g of EDTA in water. Adjust the pH with 10 N NaOH. Autoclave the solution after the preparation. This solution can be stored at RT and in darkness indefinitely.
• TE buffer (pH 7.6-8)
Add 10 mL of 1 M Tris-HCl (pH 8.0) and 2 mL of 500 mM EDTA (pH 8.0) to enough water to make one liter of TE buffer.
• 5 M NaCl
This solution can be stored indefinitely at RT.
• PBS 1X
Dilute 10X PBS in water. The solution can be stored at RT for some weeks.
• Hybridization buffer.
Deionize formamide as follows: add 5 g ion exchange resin beads (blue color) per 100 mL of formamide; stir for 1 h (resin will turn to yellow color); filter the formamide using Whatman or Millipore paper. For long-term storage aliquot deionized formamide at -20°C.
To make hybridization buffer add the different components as follows: 10 mL of deionized formamide, 12 μL of 5 M NaCl, 400 μL of 1 M Tris-HCl (pH 8.0), 20 μL of 0.5 M EDTA (pH 8.0), 2 g of dextran sulphate, 400 μL of 50X Denhart´s solution and 1 mL of 10 mg/ml herring sperm DNA. Add water to obtain 20 ml final volume. Stir the solution at least for 1 h at RT. For long-term use this solution can be stored at -80°C.
CAUTION. Formamide is toxic and flammable.
CRITICAL. Prepare this solution under RNase-free conditions.
• Probe solution
DNA probe, dissolved in water, can be stored aliquoted at -20°C in brown colored 1.5 ml tubes. Denature the probe (1:100) at 80°C for 5 min. Immediately put it on ice before adding it to the hybridization buffer.
CRITICAL. Use the solution immediately.
• PBS + Triton (PBT)
To obtain 40 ml solution of PBT, dissolve 120 µl of Triton X-100 in 39.880 ml of 1X PBS.
• Blocking solution
Add BSA to have 0.5% final concentration and donkey serum to have 5% final concentration in PBT solution. This solution can be stored for a few days at 4°C.
• Preabsorbed primary antibody solution.
To reduce unspecific background, sheep anti-Muscleblind antibody was preabsorbed against Drosophila embryos (0-6 h old; any genotype is valid because the protein is not expressed in young embryos).
Add 1 mL of blocking solution containing Muscleblind antibody diluted 1:200 to an 1.5 ml tubes containing the Drosophila fixed embryos. Leave it overnight at 4ºC with gentle agitation (e.g. rocker) and remove the supernatant containing the antibody solution without embryos. Preabsorbed antibodies can be stored for some days at 4°C.
• Secondary antibody solution.
Dilute the antibodies in blocking solution (1:200). Use these solutions immediately.