488 click it kit imaging kit

Embryos and larvae are injected with a microcapillary needle in the intestinal region (either directly into the intestine or peritoneal space) with either 2.5 mM 5-ethynyl-2'-deoxyuridine (EdU) or 50 mM 5-Bromo-2'-deoxyuridine (BrdU). EdU or BrdU injected individuals were grown to indicated time. At end of the period, each group was fixed in 4% formaldehyde, 2 h to overnight. Incorporation of EdU was detected following the standard protocol for the 488 Click-it Kit imaging kit (Invitrogen). For detection of BrdU, individuals were exposed to 0.2 N HCL for 30 minutes at room temperature followed by anti- BrdU antibody (Sigma) incubation overnight at 4° C. Secondary antibody is Alexa Fluor 594-conjugated anti mouse IgG (1:500) (Molecular Probes-Invitrogen). Previously, control single labeling of EdU or BrdU was processed for detection of the opposite nucleotide (injected at 4 dpf and grown to 5 dpf) (Li et al., 2019 ). While the injected nucleotide was detected, there was no cross-reactivity for the opposite nucleotide demonstrating the specificity of EdU and BrdU detection.

Embryos and larvae are micro-injected with a micro-capillary needle in the intestinal region (either directly into the intestine or peritoneal space) with either 10 mM 5-ethynyl-2′-deoxyuridine (Edu) or 50 mM 5-bromo-2′-deoxyuridine (Brdu). For pulse experiments, Edu or Brdu injected individuals were grown 15 to 60 min following injection to allow for nucleotide incorportation. For chase experiments, individuals were injected with either nucleotide and then grown to the described time. At end of the period, each group was fixed in 4% formaldehyde 2 hr to overnight. Incorporation of Edu was detected following the standard protocol for the 488 Click-it kit imaging kit (Invitrogen). For detection of Brdu individuals were exposed to 0.2 N HCl for 30 min at room temperature followed by anti-BrdU antibody (Sigma) incubation overnight at 4°C. Secondary antibody is Alexa Fluor 594-conjugated anti-mouse IgG (1:500) (Molecular Probes-Invitrogen).