Ultra cell conditioning 1

From individual formalin-fixed paraffin-embedded tissues, 3 mm diameter cores were taken and then placed in fresh paraffin blocks from the recipient. Analysis focused on two tissue cores from the most representative tumor areas. Immunohistochemistry (IHC) was conducted on 4 μm thick paraffin sections utilizing a BenchMark ULTRA from Ventana Medical Systems Inc. (Oro Valley, AZ, USA) and the Optiview DAB IHC Detection Kit (Ventana Medical Systems Inc., Oro Valley, AZ, USA). Polyclonal antibodies specific to Snail (1:750) and monoclonal antibodies for ERK3 (Cambridge, UK, 1:50) were applied for IHC. ULTRA Cell Conditioning 1 (Ventana Medical Systems Inc., Oro Valley, AZ, USA) served for antigen retrieval for Snail and ERK3, with a primary antibody incubation time of 32 min.

IHC staining was performed using the Discovery Ultra autostainer (Ventana, Roche, Tucson, AZ) on formalin-fixed paraffin-embedded whole tissue sections. The slides were deparaffinized in the instrument (68°C, 3 cycles á 12 min). Antigen retrieval was applied using ULTRA Cell Conditioning-1 (Ventana, Roche) for 32 minutes at 95°C. The sections were incubated for 32 minutes with IVD primary mouse monoclonal antibody anti-CD34 (clone QBEnd/10; cat# 790-2927; Ventana, Roche). As a secondary antibody, OmniMap anti-Mouse HRP (cat# 760-4310, Ventana, Roche) was loaded for 16 minutes, followed by 4 minutes of HQ HRP amplification (cat# 760-052, Ventana, Roche). The detection chromogen used was the DAB kit (cat#760-159; Ventana, Roche) with 32 minutes of incubation. Counterstaining was performed using hematoxylin II (cat#790-2208, Ventana, Roche) for 12 minutes and then loading bluing reagent for 4 minutes.
Slides were digitized using a Pannoramic 250 Flash III (3DHistech, Budapest, Hungary), slide scanner.