Alexa fluor 488 conjugated goat antirabbit antibody

Cells were cultured on coverslips, and fixed with 4% formaldehyde. The recommended protocol of antibody manufacturer (Cell Signaling) was followed. The cells were incubated with the primary antibodies E-cadherin (Cell Signaling, 3195), Slug (Cell Signaling, 9585), fibronectin (Sigma, F7387), vimentin (Cell Signaling, 5741) at 4 °C overnight and probed with Alexa-Fluor 488-conjugated goat antirabbit antibody (Cell Signaling, 4412) for fluorescence detection. The images were visualized under EVOS Imaging System (ThermoFischer, U.S.A.).

Frozen sections of tissues were cut (7μm), mounted on Superfrost Plus Gold slides and fixed with 3% paraformaldehyde for 15 mins at RT. Sections were blocked with 5% normal goat serum (Vector Laboratories)/0.3% TX-100/PBS for 60 mins at RT and incubated with anti-p-histone H2A.X (1:100) in 1% normal goat serum/0.3% TX-100/PBS o/n at 4°C. AlexaFluor® 488-conjugated goat-anti-rabbit antibody (Cell Signaling; cat. #4412) was used as secondary antibody (1:50, 60 mins, RT). After mounting with Vectashield Mounting Medium containing propidium iodide (Vector Laboratories), immunofluorescent sections were assessed by confocal laser microscopy (Axiovert 100M with laser scan head LSM 510; Carl Zeiss, Jena, Germany). The multitrack option and sequential scanning for each channel were used to eliminate any cross talk of the chromophores. As positive control for p-histone H2A.X-labelled cells, frozen jejunal sections from TLR2 KO mice treated with methotrexate were used [36 (link)].