Immunohistochemistry for ELOVL5 and Ki67 in Breast Cancer

Paraffin-embedded tumor breast tissue samples from breast cancer patients with or without metastasis were deparaffinized and rehydrated. Tissue sections were saturated with peroxidase blocking buffer and incubated with a primary antibody against ELOVL5 (#HPA047752; Sigma-Aldrich) for 1 h. After washing, HRP polymer-conjugated secondary antibodies (K4002; Dako) were then incubated with the tissue sections for 20 min followed by a 5-min coloration with 1 g/ml of DM827-3, 3’-diaminobenzidine (DAB). Hematoxylin and eosin staining was used to visualize nuclei. Tissue sections were mounted with an organic mounting solution from Dako. Quantification of ELOVL5 staining was analyzed with QuPath 0.2.3 [50 (link)] after slides were scanned by Nanozoomer controlled by NDP.view scan software (Hamamatsu). Data are presented as the average H-score of ELOVL5 expression in each slide comparing tumor and adjacent non-tumoral tissues.
For Ki67 staining, the protocol is as above using a primary antibody against Ki67 (#MIB-1 clone; Agilent) and an anti-mouse HRP polymer-conjugated secondary antibody (K4000; Dako). The H-score correlations between ELOVL5 and Ki67 are demonstrated by comparing the tumoral matching zones of the same patient.