Alexa fluor 594 dye

For Fluorescence in situ hybridization (FISH) labeling, LINC00273 probe (GenePharma) was fluorescence‐labeled using FISH Tag™ RNA Red Kit with Alexa Fluor® 594 dye (red; Invitrogen). A549 cells, which had been transfected with pcDNA3.1/LINC00273, were incubated with denatured probe (at 80℃ for 2 min) in hybridization buffer at 55℃ overnight. Next, for immunofluorescence labeling, cells were rinsed with PBS, immobilized with paraformaldehyde, permeabilized with Triton X‐100 and blocked with 1% bovine serum albumin (BSA; Sigma‐Aldrich) for 30 min. Subsequently, cells were incubated with NEDD4 antibody (Invitrogen), 1% BSA, and 0.1% Tween 20 (Sigma‐Aldrich) in PBS at 4℃ overnight. Alexa Fluor® 488‐conjugated goat polyclonal anti‐rabbit IgG (green; Abcam) secondary antibody was then applied at 37℃ for 1 h of incubation. DAPI was used for nuclear staining. Cells were observed using a Leica DMi8 inverted microscope (Leica Microsystems).

Pax7-ZsGreen cells were isolated by FACS and plated into 0.1% gelatin-coated 96-well plates (1,000 cells/well) containing muscle growth medium (MGM) with 20% charcoal-stripped (CS) FBS (NB036790; Thermo Fisher Scientific). The cells received MGM with or without E₂ daily (100 pM final concentration; E8875; Sigma-Aldrich). On day 6, Click-iT EdU cell proliferation kit for imaging, Alexa Fluor 594 dye (C10339; Invitrogen) was performed according to the manufacturer’s instructions. The cells were then incubated in DAPI (1:1,000 dilution) in PBS for 20 min at room temperature. EdU+ nuclei were identified and imaged at ×10 magnification, taken on a Zeiss Observer.Z1 inverted microscope equipped with an AxioCam MRm camera (Thornwood, NY).

All confocal images were acquired by Zeiss Zen software on a Zeiss LSM780 confocal laser scanning microscope (Carl Zeiss, Munich, Germany) with the following objectives: Plan-Apochromat 10× (NA of 0.45, air) and Plan-Apochromat 40× (NA of 1.30, oil). The following fluorochromes were used: Alexa Fluor 488 Dye (Invitrogen), Alexa Fluor 594 Dye (Invitrogen), and DAPI (Invitrogen). Immunohistochemistry images were taken with a Leica DC300F digital camera attached to a Leica DMLB microscope (Leica, Wetzlar, Germany). Images were viewed and adjusted with brightness and contrast by Adobe Photoshop software. Fluorescent images were processed and analyzed in a pipeline created in the BioimageXD framework (Kankaanpää et al., 2012 (link)). The pipeline detects the nuclei from the Hoechst channel and counts them. The cytoplasm of each cell was modeled with a 20-pixel ring around the nucleus. To quantify the invasion of neutrophils and macrophages in the colon tissues, sectioned samples embedded with paraffin from three different mice, and different time points per genotype were immunohistochemically stained for neutrophils (MPO) and macrophages (CD11b). Positively stained cells with distinct cellular borders and all cells presented within the sectioned samples were counted via a Zeiss Axiophot light microscope.

After transfection for 48 hours, transfected melanoma cells were collected for flow cytometric analysis via the Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 594 dye (Invitrogen) based on the directions. FACSCalibur flow cytometer (FACScan®; BD Biosciences) with FlowJo V10 (BD Biosciences) was employed for detection. Samples were divided into phases, and the proportion of dying, prelethal, and apoptotic cells was assessed to the standard within every trial.

A Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection using Alexa Fluor 594 dye (Thermo Fisher Scientific), and a PCNA assay (DAKO), were performed on paraffin-embedded sections according to the manufacturers’ protocols. After double staining with anti-CD31 antibody and either TUNEL or PCNA to detect apoptotic or proliferative ECs, respectively, the numbers of TUNEL- or PCNA-positive ECs was quantified from 40x magnification fields (n = 12 microscopic fields for TUNEL and PCNA assays, n = 3 animals/group).