Peg 8000 mw

Essentially following the protocol of Kuwajima et al., 2013 (link), Development
After post fixing the immunostaining all steps in the dark:

Remove PBS from samples

Add 25% formamide/10% PEG, 1 hour rocking RT

50% formamide/20% PEG, 1 hour rocking RT

50% formamide/20% PEG, O/N at 4°C

The embryos are ready for mounting and imaging.
Caution : Do not store the embryos in formamide. For storage rinse the sample in PBS and store in fresh PBS (azide can be added).
Solutions :

50% formamide/20% polyethylene glycol (PEG): mix formamide (99.6%, considered 100%) with 40% PEG/H2O (wt/vol) at a ratio of 1:1 (vol/vol).

25% formamide/10% PEG: mix 50% formamide plus 20% PEG/H2O (wt/vol) at a ratio of 1:1 (vol/vol).

40% PEG solution: stir powdered PEG 8000 MW (Sigma-Aldrich) in warm H2O for 30 minutes, (stable at room temperature for several months)

Reverse-phase HPLC purified RNA oligonucleotides r(G₂C₄)₄ and r(G₄C₂)₄ were purchased from Eurogentec (Seraing, Belgium). After addition of 5 mL of 2 M LiCl samples were heated to 90 °C for 5 min. Samples were extensively dialyzed against autoclaved H₂O through four 30 min cycles and then concentrated using an Amicon ultrafilter (Merck Millipore, Herfordshire, UK). Following the overnight lyophilization, samples were diluted in 90% of autoclaved H₂O and 10% of ²H₂O. The concentrations of the 300 μL NMR samples were 0.9, 0.7 and 0.3 mM for r(G₂C₄)₄ and 1.1, 0.7, 0.3, 0.2, 0.1 and 0.05 mM for r(G₄C₂)₄. In the case of equimolar mixing experiments, equimolar quantities of r(G₂C₄)₄ and r(G₄C₂)₄ were diluted to final oligonucleotide concentration of 0.2 mM per sense and antisense strand. Samples of r(G₄C₂)₄ and mixed RNA oligonucleotides were also heated to 90 °C for 5 min prior to annealing. For molecular crowding experiments, sample of r(G₂C₄)₄ and mixed RNA oligonucleotides were diluted in 10% w/v PEG (8000 MW) (Sigma Aldrich, Munich, Germany) in autoclaved H₂O to 0.3 and 0.2 mM oligonucleotide concentration per strand, respectively. The pH of samples was adjusted by the addition of 0.1 mM LiOH or HCl.