Nanoliter 2000 volume microinjector

One day after oocyte isolation, individual oocytes were injected with either 25 nL of mUT-B, mUT-A2 or mUT-A3 cRNA (concentration 1 µg/µl), or an equivalent volume of sterile water. All injections were performed using an injection needle pulled with a Model P-97 Flaming/Brown micropipette puller (Sutter Instrument Company, Novato, CA, USA). Prior to use, the tips of the injection needles were aseptically cut to produce a tip that was approximately 2 μm in diameter (Kabutomori et al., 2018 (link); Musa-Aziz et al., 2010 (link)). Injections were performed with mineral oil (part #M5904, Sigma-Aldrich) filled needles placed onto a Nanoliter 2000 volume microinjector (World Precision Instruments, WPI, Sarasota, FL, USA). All cRNA-injected and H₂O-injected oocytes were stored in OR3 media, at 18°C. Routinely, the protein expression and function experiments below were performed 4 days after injection.

One day after isolation, oocytes were injected with 25 nl cRNA [25 ng (given as 25 nl of a 1 ng/nl cRNA solution)] encoding for hAQP1, hAQP1_FLAG, hAQP1_C189S mutant, rAQP3, hAQP7, hAQP8 or rAQP9 or an equivalent volume of sterile water (called ‘H₂O-injected control oocytes’). Sterile injection needles were made using a Model P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Company, Novato, USA), as previously described (Musa-Aziz et al., 2010 (link)), and aseptically cut to have a diameter of approximately 2 μm. The needles were attached to a Nanoliter 2000 volume microinjector (World Precision Instruments, Sarasota, USA), filled with mineral oil and then filled with cRNA. Oocytes were injected with 25 nl cRNA or sterile water and stored at 18°C in OR3 medium. Expression and function were evaluated 3-5 days following injection.