Anti rfp 5f8

All tissue samples were fixed with 4% paraformaldehyde overnight, decalcified with pH 7.2 EDTA buffer (G-Chelate Mild, GenoStuff, Tokyo, Japan) for 10 d at 4 °C, and embedded in paraffin. The samples were cut into 3–4-μm-thick sections. Hematoxylin and eosin, safranin O/fast Green, and Masson trichrome staining were applied using standard protocols. For immunohistochemistry, the antibodies used were anti-GFP (EPR14104) (Abcam; dilution 1:100), anti-GFP (4B10) (Cell Signaling Technology; dilution 1:200), anti-RFP (5F8) (Chromotek; dilution 1:200), anti-tenomodulin (Thermo Fisher Scientific; dilution 1:150), anti-αSMA (1A4) (Abcam; dilution 1:200), and anti-bFGF (Bioss; dilution 1:200). For DAB staining, the secondary antibody used was Dako EnVision (Dako Japan Inc., Kyoto, Japan); stained cells were analysed by microscopy (BX51, Olympus). For immunofluorescence, the secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific); stained cells were analysed by fluorescence microscopy (IX83, Olympus).

50 or 100 L2 larvae in 30μL of water were flash-frozen in liquid nitrogen. 15μL of reducing or non-reducing sample buffer was added and samples incubated at 85–95°C for 10 minutes. Protein amounts were verified by Ponceau stain of membranes. Anti-RFP (5F8, ChromoTek, Germany) was used to detect DAF-28(R37C)::mCherry. JT191 and TU3401 strains were used as negative and positive controls. For the blot in S3 Fig, full plates were collected and frozen in aliquots. Worms were lysed by mechanical disruption, as described in [8 (link)], treated with reducing/non-reducing sample buffer, and further processed as above.

Section and whole-mount immunohistochemistry were performed essentially as previously described^{40 (link)}. Briefly, 5 µm paraffin or 10 µm OCT embedded tissue sections were incubated over-night at 4 °C with the following antibodies at the concentrations below. Antigen retrieval was performed only when LoxL3 antibody was used. Wholemount immunostaining was carried out essentially with the same primary antibody concentrations although incubation periods were extended to 2 weeks in 4C (for both primary and secondary antibodies). The following antibodies were used: anti-Myosin (A4.1025, 1:300; DSHB); anti-GFP (A6455, 1:500; Invitrogen); anti-RFP (5F8, 1:500; Chromotek); anti-LOXL3^{12 (link)} (1:100).
All assays were repeated at least at three time points using different cell cultures or tissue sections from distinct embryos/mice.