Diva decloaker 10x

Formalin-fixed and paraffin-embedded CRC tissues from patients with CRC were obtained from the Department of Pathology of the County Hospital Ryhov, Jönköping, Sweden. Among the eight patients, six had tumors localized in the colon and two in the rectum; these were classified as stage I in two cases, stage II in two cases, stage III in two cases, and stage IV in two cases. The staining was performed on 3.5 μm sections of paraffin-embedded tissue samples to detect the CCL4 protein expression. In brief, antigen retrieval was finished by cooking at 110°C for 52 min in Diva Decloaker, 10X (Biocare Medical, Concord, CA, United States). The slides were treated with hydrogen peroxide for 5 min to prevent the occurrence of endogenous peroxidase, which might alter the interpretation of the color. Primary rabbit polyclonal CCL4 antibody against human CCL4 (Abcam, Tokyo, Japan) was used at a dilution of 1:300. The antibody was applied to the tissue sections, which were then incubated for 30 min at room temperature. The MACH 4 Universal HRP-Polymer Detection kit (Biocare Medical) was used, and the reaction was visualized using Betazoid DAB Chromogen Kit (Biocare Medical). Human kidney tissue was used as a positive control for CCL4 expression and rabbit IgG, polyclonal isotype control (Abcam) was used as a negative control and included along with each patient tissue section.