Goat anti mouse alexa 568

Whole-mount immunostaining was performed as described previously^{69 (link)}, with the following modification: Larvae were fixed in 4% PFA after stopping the heart with 0.2% Tricaine to prevent cardiac collapse during fixation. The primary antibodies used in this study are Mouse anti-MHC (MF20) (1:500, Invitrogen 14-6503-82), Mouse anti-Alcama (1:500, ZN-8, DSHB), and Rabbit anti-Fli1 (1:500, Abcam ab133485), followed by the secondary antibodies: Goat anti-Mouse-Alexa568 (1:500, Invitrogen A11004), Goat anti-Mouse-Alexa647 (1:500, Invitrogen A21236), and Goat anti-Rabbit-Alexa488 (1:500, Invitrogen A110034). All images were analyzed using an LSM 700 confocal laser scanning microscope (Zeiss) with a 40x objective, except for the samples shown in Fig. 2t, u, which were imaged using an LSM 800 Examiner confocal laser scanning microscope (Zeiss) with a 40x objective. For AV cell number quantification, the cells were counted in every plane of a z-stack covering the AV canal. The 78 hpf wild-type dataset (n = 61) is the same in Figs. 1n, 2s, 3o, and represents a pool of pkd1a^+/+, pkd1a^+/+; pkd2^+/+, and camk2g1^+/+; camk2g2^+/+ larvae. The 78 hpf pkd1a mutant dataset (n = 26) is the same in Figs. 1n, 2s, 3o.

For immunostaining of C. elegans embryos, embryos were dissected from adults in 10 µl MilliQ HR₂RO on slides coated with poly-L-lysine. Samples were then freeze-cracked and fixed in methanol for 5 min. at −20˚C and subsequently in acetone for 5 min. at −20˚C. Embryos were then rehydrated in phosphate buffered saline +0.05% Tween-20 (PBST), blocked for 1 hr at 4˚C in PBST +1% bovine serum albumin and 1% goat serum (Sigma-Aldrich), and then incubated at room temperature with primary antibodies for 1 hr and then with secondary antibodies for 45 min., both in blocking solution, with four 10 min washes in PBST following each antibody mix. Finally, embryos were embedded in ProLong Gold Antifade with DAPI. Primary antibodies used were mouse anti-LIN-5 [1:10, (Lorson et al., 2000 (link)), mouse anti-FLAG M2 (Sigma-Aldrich) and rabbit anti-DHC-1 (1:100 (Gonczy et al., 1999 (link)); a kind gift from P. Gönczy). Secondary antibodies used were goat anti-rabbit Alexa-488, goat anti-rabbit Alexa-568, goat anti-mouse Alexa-488, goat anti-mouse Alexa-568 (Invitrogen) and goat anti-mouse Atto 647N (Sigma-Aldrich), all at 1:500 dilution in blocking solution. Imaging of immunolabeled embryos was performed on the spinning disk setup described above.

Each cell line was seeded into wells in 96-well plates one day before the assay. Cells were fixed with 4% paraformaldehyde for 1 h at room temperature and blocked by 2% (w/v) bovine serum albumin in phosphate-buffered saline (PBS) with 0.5% Tween (PBST) for 1 h at room temperature. The cells were then incubated with the primary antibody, binding either to the ACE2 [rabbit anti-ACE2 polyclonal antibody (1:500) (Abcam, USA)] or TMPRSS2 [mouse anti-TMPRSS2 monoclonal antibody (1:50) (Santa Cruz Biotechnology, USA)] proteins, at 37 °C for 1 h. After that, the cells were washed with PBST three times, and incubated with the secondary antibody; goat anti-rabbit Alexa 488 (1:500; Invitrogen, USA) or goat anti-mouse Alexa 568 (1:500; Invitrogen, USA). After washing, Hoechst dye (Thermo Fisher Scientific, USA) was used to stain cells’ nuclei. The fluorescent signals were detected by BioTek Cytation 7 Cell Imaging Multi-Mode Reader (Agilent Technologies, USA).

Antibodies used in this study were as follows: rabbit anti-GFP (Invitrogen, 1:1000), mouse anti-GFP (Invitrogen, 1:250), mouse anti-rat CD2 (AbD Serotec, 1:200), rabbit anti-pMad (gift from Ed Laufer, 1:1000), goat anti-rabbit Alexa488 (Invitrogen, 1:1000), goat anti-rabbit Alexa568 (Invitrogen, 1:1000), goat anti-mouse Alexa488 (Invitrogen, 1:1000) and goat anti-mouse Alexa568 (Invitrogen, 1:1000). Larvae were filleted, fixed in 4% paraformaldehyde for 30 minutes and then stained according to a standard protocol [69 (link)].