Mabs1262

Cells were fixed using 3.7% formaldehyde (Sigma Aldrich) and processed as described previously [17 (link)]. Resulting coverslips were probed using primary antibodies (pZIP7 at 1:100, MABS1262, Merck Millipore; ZIP6 at 1:100, in-house antibody; pHistoneH3^S10 1:100, No. 9706S, Cell Signalling) for 1 h before incubation with Alexa Fluor secondary antibodies (goat anti-mouse Alexa Fluor 594, #A11032 Invitrogen; goat anti-mouse Alexa Fluor 488, #A10684, Invitrogen; goat anti-rabbit Alexa Fluor 594, #A11072, Invitrogen) at 1:1,000 dilution for 30 min. Coverslips were mounted on microscope slides using Vectorshield^® mounting medium containing DAPI (Vector Laboratories), prior to sealing with clear nail varnish. Slides were subsequently imaged using a 63× oil immersion lens on a Leica RPE automatic microscope. Image acquisition was through use of Openlab software for Macintosh overall survival (OS) and resulting images processed using FIJI software.

Cells were fixed with 3.7% formaldehyde (Sigma Aldrich) and processed as previously described.15 (link) Cells were probed for 1 hour with primary antibody (anti-pZIP7 1/100, MABS1262, Merck Millipore, USA) followed by goat anti-mouse Alexa Fluor 594 secondary antibody at 1/1000 dilution (Molecular Probes, Invitrogen, UK) for 30 minutes. Coverslips were mounted on microscope slides with Vectorshield mounting medium with DAPI (Vector Laboratories, USA) and sealed with nail varnish. Cells were visualised on a Leica RPE automatic microscope using a 63× oil immersion lens. Pictures were acquired and processed using Openlab software for Macintosh operating system.

Samples of MCF-7 and TAMR cells were fixed in 3.7% formaldehyde before incubation with pH 8 EDTA buffer in a pressure-cook microwave for 2 min at 950 W for optimal antigen retrieval. This was followed by blocking with serum-free blocking reagent (DAKO) and incubation with mouse monoclonal pZIP7 antibody (MABS1262, Merck Millipore) at 1:8,000 dilution for 1 h at room temperature in a humidity chamber. Slides were then briefly washed with phosphate buffered saline/Tween and incubated with secondary antibody [Mouse Envision labelled polymer-horse radish peroxidase (HRP), No. K4001, DAKO] for up to 1 h. 3’-3’-diamobenzidine (DAB) chromogen-substrate solution (DAKO) was used to visualize target protein and 0.05% (aqueous) methyl green was used to counterstain nuclei. Images were obtained using Olympus BH-2 microscope with multiple fields of view being imaged for analysis.