Low ph target retrieval solution

The initial core biopsy performed at the time of diagnosis was analysed for Ki-67 as were the repeat biopsies after the first cycle of NAC where available. Four-micron-thick sections were cut onto glass slides (Thermo Scientific Superfrost Plus, Braunschweig, Germany). After drying at 37 °C overnight, the sections were dewaxed in xylene and then rehydrated by immersing in decreasing concentrations of industrial-methylated spirits (IMS). The sections were then washed in tap water. Antigen retrieval was performed by microwaving the sections at 800 W for 10 min in preheated Dako low-pH Target Retrieval solution pH 6. Staining was conducted in a Dako Autostainer (Dako, Glostrup, Denmark) using the Dako REAL Detection System. Staining was performed using the MIB 1 primary antibody (Dako) at a dilution of 1 in 50. At the end of the autostainer run, the sections were removed from the instrument and counterstained with Mayer's Haematoxylin, dehydrated in increasing concentrations of IMS, cleared in xylene and mounted in DPX. A known positive Ki-67 control tissue was stained at the same time as the test sections, and in addition was run without primary antibody as the negative control. However the poor quality of the repeat biopsies performed after the first cycle of NAC did not allow further evaluation of this material.

Paraffin sections were immunostained by Dako Autostainer using Dako Envision™ FLEX+ system (K8012; Dako, Glostrup, Denmark) as described in our previous study (15 (link)). Briefly, the sections were deparaffinized, epitopes were unmasked in PT-link with low pH target retrieval solution (Dako) and then blocked with peroxidase blocking solution (Dako) for 5 min. The slides were incubated at room temperature with polyclonal rabbit anti-human ZEB1 antibody (cat. no. HPA027524; 1:500; Sigma-Aldrich, St. Louis, USA), followed by incubation with rabbit linker for 15 min and horseradish peroxidase for 30 min at room temperature. The slides were subsequently stained with 3,3′-diaminobenzidine tetrahydrochloride for 10 min and counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Fisher Scientific, Waltham, MA, USA). A known ZEB1-positive human esophageal carcinoma slide was used as positive control. Serial negative controls were tested by the same concentration of normal rabbit serum as a substitute for the rabbit anti-human ZEB1 antibody.