Click it edu imaging kit with alexa fluor 488

The Click-iT EdU imaging kit with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) was used for EdU (5-ethynyl-2′-deoxyuridine) imaging analysis. We added 1 mM EdU to the suspensions of synchronized cells 1 h before the dark period. To enhance the incorporation of EdU to cpDNA, the thymidine pool in chloroplasts was depleted by adding 1 mM FdUrd (5-fluoro-2′-deoxyuridine)^{16 (link),17 (link)}. After a 4-h incubation, the cells were fixed with 3.7% formaldehyde for 10 min on ice. Cells were collected by a centrifugation at 3000 r.p.m. for 1 min, washed twice with ice-cold wash buffer (3% bovine serum albumin and 10 mM HEPES (pH 6.8)), and then permeabilized with a buffer containing 5% Triton, 3% bovine serum albumin, and 10 mM HEPES (pH 6.8) for 20 min on ice. After washing twice with ice-cold wash buffer, the precipitated cells were suspended in the Click-iT reaction mix and incubated for 30 min on ice. Samples were not incubated at room temperature to avoid the non-specific staining of oil bodies. Cells were washed twice with the wash buffer, co-stained with DAPI, and observed with the BX51 microscope.

Cells were seeded at 0.2 × 10⁵/well in 12-well plates and treated with the indicated compounds and controls overnight. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU, 10 μM) was analyzed following addition for 4 h according to the manufacturer’s protocol. Detection of EdU was performed using Click-iT EdU Imaging Kit with Alexa Fluor 488 (Invitrogen). DAPI (1 μg/ml) was used for labeling of nuclei. Images were acquired using an Echo Revolve fluorescence microscope at 20X magnification. The total number of EdU+ cells was determined using 10 representative fields, and the rate of proliferation was calculated as percentage of EdU^+/DAPI.

For EdU detection, the Click-iT EdU Imaging Kit with Alexa Fluor 488 (Thermo Fisher) was used. The stock solutions were prepared according to the provided protocol included in the kit (5 mg/ml EdU stock solution). For EdU incubation, a fresh working solution of 10% DMSO/50 µg/ml EdU diluted in E3 water was prepared in 1.5 ml Eppendorf tubes containing a total volume of 1 ml. Fish were set-up in pairs and eggs were collected either directly after laying or after 45 mpf. After collection the eggs were transferred into the EdU solution using a transfer pipet and incubated for 30 min on ice in the dark. The embryos were then transferred to a 28°C incubator to incubate for 30 min. After incubation, the embryos were transferred into fresh E3 water for a short rinse and directly fixed after with microtubule fix (3.7% formaldehyde, 0.25% glutaraldehyde, 5 mM EGTA and 0.3% Triton X-100) for 2 h at room temperature as described below in the ‘Fixation and antibody labeling’ section. Detection of EdU was performed according to the protocol described in the Click-iT EdU Imaging Kit. The imaging of the EdU detection was performed as described in the ‘Imaging’ section below.