Goat anti mouse atto 647n

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.

Immunofluorescence stainings in nephrocytes were performed as described before.¹⁹ (link) In brief, nephrocytes were dissected in phosphate-buffered saline followed by fixation in 4% formaldehyde for 20 minutes and methanol for 1 hour at room temperature. After 3 washing steps in wash buffer (phosphate-buffered saline, 0.3% Triton, 0.5% bovine serum albumin), primary antibodies were incubated in wash buffer overnight at 4 °C. Primary antibodies were as follows: mouse antipolychaetoid 2 (Developmental Studies Hybridoma Bank, 1:25), goat anti–horseradish peroxidase (Jackson ImmunoResearch #123-005-021, 1:200), and rabbit anti–HA (Sigma #H6908, 1:100). After 3 washing steps and 30 minutes blocking in 5% normal donkey serum, the tissue was incubated with secondary antibodies (donkey anti-rabbitCy3, Jackson ImmunoResearch #711-165-152, 1:250; donkey anti-goatAlexa488, Jackson ImmunoResearch #705-545-003, 1:250; goat anti-mouseAtto647N, Sigma #50185, 1:1000) for 1 hour at room temperature. Following 3 washing steps, the tissue was mounted in Vectashield mounting medium (Linaris, H-1200).