Dkk3 antibody

Western was performed using a 1:1000 dilution of DKK3 antibody (Santa Cruz) and a 1:5000 dilution of goat anti-rabbit secondary antibody (Vector). While the calculated molecular weight of DKK3 is 38 kDa, the size on Western has been reported as 50-55 kDa in reducing conditions due to glycosylation.^{13 (link)} For SMAD4, a 1:2000 dilution of SMAD4 antibody (Abcam) was used with a 1:8000 dilution of anti-rabbit secondary antibody (Vector). ß-actin was used as a loading control with a 1:10000 dilution of ß-actin antibody (Abcam) and a 1:10000 dilution of anti-mouse secondary antibody (Vector).

Tissue microarrays were constructed with formalin-fixed, paraffin-embedded tissues from 73 patients including 64 EAC, 8 dysplastic Barrett's mucosa, 11 BM, and 2 normal esophageal samples.¹⁴ Immunohistochemical staining was done on the DAKO Autostainer using DAKO LSAB+ and 3,3′-diaminobenzidine as the chromogen. Dewaxed and rehydrated sections were labeled with DKK3 antibody (1:200 dilution; Santa Cruz). Microwave citric acid epitope retrieval was performed for 20 minutes. Slides were counterstained with hematoxylin. Samples were scored using a scale of 0 (no staining), 1+ (<10% staining), 2+ (10-50%) staining, or 3+ (≥50%).^{15 (link)} The scoring was repeated to ensure reproducibility.