Three channel tcs sp2 laser scanning confocal microscope

Cells fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, saturated with 0.5% serum albumin in PBS and incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-AhR and rabbit polyclonal anti-AhRR (Abcam, UK). Chicken anti-rabbit Alexa Fluor 633 or chicken anti-mouse Alexa Fluor 488 labelled were used as secondary antibodies. Nuclei were counterstained with Propidium Iodide.
Images were collected by a three-channel TCS SP2 laser scanning confocal microscope (Leica Wetzlar, Germany). Co-localization was analyzed through two-dimensional correlation cytofluorograms accomplished by macro routines integrated as plugins in ImageJ 1.4 software (Wayne Rasband, NIH, Bethesda, MD, USA).

To assess apoptosis in living cells, HUVECs were seeded in 8-well chamber slide (IBIDI®, Gräfelfing, Germany) at the density of 100x10³ cells/well. Apoptosis detection Kit (Dojindo Molecular technology Inc. Rockville, MD, USA) was used according to the manufacturer’s protocol. Briefly, at the end of treatments cells were washed, and incubated with FITC-Annexin V (1:20) plus propidiun iodide (PI, 1:20) in the supplied buffer for 10 minutes. Then, cells were washed and immediately observed using a three-channel TCS SP2 laser-scanning confocal microscope (Leica, Wetzlar, Germany), equipped with 458, 476, 488, 514, 543 and 633 nm excitation lines.
To acquire images also of fixed cells, FITC-Annexin V Apoptosis detection Kit (BioVision Research, USA) was used according to the manufacturer´s protocol. HUVECs were seeded on 8-well chamber slides (Nalge Nunc International) at the density of 100 × 10³ cells per well. At the end of the treatments, cells were incubated for 10 min in the dark with Annexin V-FITC (1:100) and then with DAPI solution (1:1000) for the same time. Subsequently, HUVECs were fixed in 2 % PFA before visualization. Images were acquired using epifluorescence microscope (Olympus 1x81) with a 60X oil immersion objective.

Mock cells (either NMuMG or 4T1) or their stable transfectants overexpressing FLAG-tagged METTL7A1 were plated on glass chamber-slides (60 000 cells/well). Immunofluorescence was carried out 2 days after plating essentially as reported in (1 (link)). Rabbit polyclonal anti-METTL7A antibody (Abcam) and anti-FLAG antibody (F1804, Sigma) were used at a 1:500 dilution. Secondary antibodies, Alexa Fluor 488 anti-rabbit (Thermo Fisher) dilution 1:400, Alexa 488 anti-mouse (ThermoFisher) dilution 1:400, were diluted in IF buffer and incubated for 30 min at 37°C. ER Tracker dye for endoplasmic reticulum labeling was from Molecular Probes (E34251). Nuclei were counterstained with TO-PRO-3 (ThermoFisher). Images were collected using a three-channel TCS SP2 laser-scanning confocal microscope (Leica Microsystems). Images were imported into ImageJ to split and merge channels, cropped and adjusted for resolution and for intensity level range using Photoshop (scale bar = 10 μm).