Tl led microscope

glass capillaries (Harvard Apparatus glass capillaries with 780 μm inner diameter) are pulled using a needle puller and microforge to build a holding pipette and an injection needle. The resulting injection needles are filled with RNA and or protein solution diluted in injection buffer (5 mM Tris–HCl pH = 7.4, 0.1 mM EDTA) to the following concentrations: mCherry‐EB3 100 ng/μl, Ect2‐GFP, LifeAct‐GFP, LifeAct‐RFP, Lap2b‐GFP and Cdc42T17N 200 ng/μl and Cdc42 reporter 150 ng/μl. To knock out Myo1c, zygotes were injected with 300 ng/μl Cas9 protein (IDT, 1081058) and 80 ng/μl gRNA1 and gRNA2 each diluted in injection buffer. The filled needle is positioned on a micromanipulator (Narishige MMO‐4) and connected to a positive pressure pump (Eppendorf FemtoJet 4i). Embryos are placed in FHM drops covered with mineral oil under Leica TL Led microscope. Zygote or 2 cell‐embryos were injected while holding with holding pipette connected to a Micropump CellTram Oil.