Click it alexa fluor 647 imaging kit

For EdU pulse-labeling, 1 μl of 2.5 mg/ml Edu solution from the Click-iT Alexa Fluor 647 imaging kit (Life technologies, Cat# C10340) was injected retro-orbitally into anesthetized transgenic fish at 5 weeks of age. Two hours after injection, the fish were fixed for cryosectioning according to the protocol below, with immunostaining according to the manufacturer's protocol.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using the Click-iT Alexa Fluor 647 Imaging kit (Thermofisher) according to manufacturer’s instructions on tissue sections from control and DT-treated Vegfr3-CreERT2 iDTR^flox/+ mice and Vegfr3-CreERT2 iDTR^flox/+ tdTomato^flox/+ mice to label cells exhibiting DNA fragmentation. Briefly, fixed tissue sections were washed and permeabilized with Proteinase K solution for 15 minutes, washed and immersed in 4% paraformaldehyde (PFA) for 5 minutes at 37°C. After that slides were washed again, rinsed in deionized water and TdT Reaction buffer was added to each slide. Slides were incubated at 37°C for 10 minutes, TdT reaction buffer was removed and TdT reaction mixture was added to each slide for a 60 minutes long incubation at 37°C. Slides were rinsed in deionized water, washed with 3% BSA and 0.1% Triton X-100 in PBS for 5 minutes and rinsed in PBS. This was followed by the TUNEL Click-iT reaction i.e. Click-iT Plus TUNEL reaction cocktail was added and slides were incubated at 37°C for 30 minutes, washed with 3% BSA in PBS and rinsed in PBS. For additional immunofluorescence stainings slides were processed as described i.e. the TUNEL Click-iT reaction was followed by blocking and incubation in primary antibodies.

At 4 dpf Tg(olig1:nls-mApple) zebrafish embryos were incubated in 0.4 mM
5-ethynyl-2’-deoxyuridine (EDU) in Danieau’s solution. After 6 h
incubation, embryos were incubated for 15 minutes in 2mg/ml Pronase (Sigma
Aldrich) and subsequently fixed for 2h in 4% PFA. Whole embryos and transverse
spinal cord sections were stained for EDU using the Click-IT™ Alexa
Fluor™ 647 Imaging Kit (ThermoFisher Scientific) as detailed in the kit
protocol, with the exception of a 1.5h ClickIT reaction incubation time.
Afterwards, immunofluorescence staining was performed for transgene
detection.