Alexa fluor 594 anti mouse

After washing with 1 x PBS, cells were permeabilised in 0.1% Triton X-100 in PBS for 5 min and blocked in 1% BSA in PBS for 30 min. Staining was performed overnight with the following primary antibodies at a concentration of 1:500: rabbit anti-γH2AX (Novus Biologicals); rabbit anti-53BP1 (Novus Biologicals), mouse anti-DNA-PKcs (Abcam), rabbit anti-TDP-43 C-terminus (405–414) (Cosmo Bio Co., LTD), and rabbit anti-phospho-TDP-43 (Cosmo Bio Co., LTD). The following secondary antibodies were used: anti-rabbit Alexa Fluor 488, (Life technologies), anti-mouse Alexa Fluor 594 (Life technologies) or anti-rabbit Alexa Fluor 647 antibodies (all Life Technologies) for 2 h. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) or DAPI (Sigma Aldrich).

For colony visualisation, cells forming colonies on glass coverslips were fixed in 4% formaldehyde, incubated for 1 h in blocking/permeabilization solution (1% BSA, 5% horse serum, 0.1% saponin in PBS) and stained with rhodamine phalloidin. Images were captured with an Olympus FV1000 microscope and Olympus Fluoview software. Channels were split and merged, and image intensity was adjusted where required using ImageJ software. For cell–cell contact analysis, cells were blocked for 1 h in 1% BSA with 5% horse serum in PBS and permeabilized with 0.1% saponin. Cells were incubated with mouse anti-EPHB6 (Santa Cruz) or a matching non-specific IgG for 72 h, rinsed three times with PBS and incubated with anti-mouse Alexa Fluor 594 (Life Technologies, # 8890) in 0.1% BSA in PBS for 1 h. Cells were rinsed and stained with anti-ZO-1 Alexa Fluor 488 or a matching non-specific IgG Alexa Fluor 488 (Thermo Fisher, #339188, #53-4714-80). ProLong Gold antifade with DAPI (Life Technologies) was used as a mounting medium in all confocal microscopy experiments. Cells were visualised with a Carl Zeiss LSM 700 confocal microscope. Z-stack frames were acquired using ZEN 2012 software.

Immunofluorescence (IF) histology was performed on 5 μm transverse cryosections. Standard protocol was used for these staining as per our previous publications (Nandi et al., 2016 (link)). The primary (1:100) and secondary antibodies (1:200) used were; HIF-1α (cat# ab179483), and Sarcomeric alpha-actinin (cat# ab9465) from Abcam, anti-mouse Alexa^®Fluor 594 (cat# A21201) and anti-rabbit Alexa^®Fluor 488 (cat# A21441) from Life Technologies, and anti-rabbit-HRP (cat# sc-2054) from Santa-Cruz Biotechnology. Images were captured using bright-field microscope (Leica Microsystems, United States) and Olympus IX71 fluorescence Imaging Systems (Olympus, United States) respectively. Images were quantified using ImageJ software (NIH, United States).