Serum free blocking reagent

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.

For zRMS studies, tumor-burdened fish were euthanized in ice-water, fixed in 4%PFA overnight at 4°C, washed in PBS for 24 h, decalcified in 20% EDTA pH 8.0 for 24 h, dehydrated in 70% EtOH, and paraffin-embedded. Paraffin-embedded tissues were cut into 10–15 µm thick sections and stained with H&E or further processed for antibody staining. For mouse xenografts following dissection, mouse tumor tissue was fixed in 4% PFA overnight, washed in PBS for 24 h, and dehydrated in 70% EtOH prior to paraffin-embedment. For all downstream IHC stains (zRMS, mouse xenograft, human tissue array), slides were de-paraffinized and retrieved in either pH6 (Six1, myHC) or pH9 (Pax7) Tris/EDTA buffer. Slides were then peroxidase blocked with 3% hydrogen peroxide (in methanol) for 10 min, blocked in serum-free blocking reagent (DAKO) and incubated with primary antibodies for 1hr at room temperature. Appropriate species’ secondary antibodies were then incubated for 30 min and developed with DAB stain for 10 min and counterstained with hematoxylin for another 8 min.