Ab227584

Immunofluorescence studies for the detection of AGR2 (1 in 100 dilution) (Abcam, Cat No: ab227584), WFDC2 (1 in 250 dilution) (Abcam, Cat No: ab200828) and TSC22D3 (1 in 50 dilution) (Abcam, Cat No: ab197987) were performed as described previously [24 (link),33 (link),34 (link)]. Epithelial expression of AGR2, WFDC2 and TSC22D3 positive cells was confirmed using cytokeratin (1 in 500) (Biocare, Denmark) and appropriate Alexa fluorophor conjugated secondary antibodies (Thermo Fisher Scientific, Austin, TX, USA). Localization of AGR2 protein expression to the endoplasmic reticulum (ER) was confirmed using the ER marker GRP94 (1 in 1000 dilution) (Abcam, Cat No: ab210960).

For immunohistochemical (IHC) analysis, 4-µm-thick tissue sections were prepared from formalin-fixed, paraffin-embedded primary tumors. The following antibodies against human proteins were used: anti-α-SMA (clone 1A4; 1:50; DAKO, Carpinteria, CA, USA), anti-CD31 (PECAM; 1:10; BioGenex, Fremont, CA, USA); anti-AGR2 (ab227584; 1:100; Abcam, Cambridge, UK), and anti-CXCR4 (ab181020; 1:100; Abcam). The tumor proliferation index was determined by counting the number of tumor cell nuclei that reacted with the MIB-1 antibody, which is specific to the Ki67 antigen. For each case, 1000 nuclei were counted. The final result is presented in terms of the percentage of labeled nuclei in the sample, representing the Ki67 index. The pathological specimens were graded as follows by two pathologists who were blinded to clinical parameters: Grade I (<2 mitoses per 10 high-power fields [HPFs]; Ki-67 index: <3%), Grade II (2–20 mitoses per 10 HPFs; Ki-67 index: 3–20%), or Grade III (>20 mitoses per 10 HPFs; Ki-67 index: >20%).