Ab115957

Bioinylated Jaclin (lectin for T-antigen, 1:15mg/ml, Vector Laboratories, U.S.A), Peanut Agglutin Jaclin (lectin for sT-antigen, 1:15mg/ml, Vector Laboratories, U.S.A), Vicia Villosa Lectin Jaclin (lectin for Tn-antigen, 1:15mg/ml, Vector laboratories, U.S.A) and anti-sialyl Tn monoclonal antibodies (1:30, Abcam, ab115957, Cambridge, MA) was applied for FFPE immunohistochemistry testing. The staining protocol was based on recommendation from Vector Laboratories (http://vectorlabs.com) and Nordic Immunohistochemical Quality Control organization (www.nordiqc.org). All of cases was stained by IHC-p at once. The staining score was evaluated by two independent pathologists without the knowledge of clinicopathological data. The expression intensity of Tn, sTn, T, sT was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) and the staining extent was scored as the percentage of positive area in all intercellular space (0-100%). The staining intensity and extent were multiplied to generate an immunohistochemistry score (H-score) on a continuous scale of 0-300. We selected the optimum cutoff score for the expression of these antigens by using X-tile software version 3.6.1 (Yale University School of Medicine, New Haven, CT, USA) based on the association with the patients’ OS.

For analysis of surface markers, T cells were collected at indicated time points after activation with CD3/CD28 Dynabeads (11131D, Thermo Fisher). For analysis of CD11b expression on T cells, activated T cells were collected at day 8, washed in Flow Cytometry Staining Buffer (00-4222-26, Thermo Fisher) and incubated with anti-CD11b biotin antibody (553309, BD Biosciences; 1:100) for 30 min at 4 °C. Cells where then washed and incubated with anti-CD3 BUV805 (612894, BD Biosciences; 1:100), anti-CD4 BUV395 (563550, BD Biosciences; 1:200) anti-CD8 APC/H7 (566855, BD Biosciences; 1:200) and streptavidin PE (12-4317-87, Thermo Fisher; 1:200) for 30 min at 4 °C in the dark. After washing, cells were resuspended in 200 µL staining buffer containing DAPI (1:10,000) (D1306, Invitrogen).
For STn surface staining, cells were incubated with anti-STn primary antibody (ab115957, Abcam; 1:100) for 30 min at 4 °C. Cells were then washed and incubated with an anti-mouse IgG-FITC secondary antibody (406001, Biolegend; 1:200) for 30 min at 4 °C in the dark. After a final wash step, cells were resuspended in 200 µL of 2 % BSA in PBS with DAPI (1:10,000). Data were collected on a FACSymphony flow cytometer and analyzed using FlowJo (BD Biosciences).

These experiments were performed as we previously described^{21 (link)–23 (link)}. For Western blot, antibodies for GAPDH (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cosmc (sc-67480; Santa Cruz Biotechnology), CEL (ab79131; Abcam, Cambridge, UK; sc-34883; Santa Cruz Biotechnology), DMBT1 (MAB59151; R&D Systems, Minneapolis, MN, USA), elastase (ab21593; Abcam) and trypsin (ab166898; Abcam) were used. Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. For immunohistochemistry, antibodies for Tn Antigen (MA180055; Thermo Fisher Scientific, Grand Island, NY, USA), STn Antigen (ab115957; Abcam), insulin (8138; CST, Beverly, MA, USA) and VVA-fluorescein (FL-1231; Vector Laboratories) were used at a dilution of 1:100^{23 (link)}. Ten micrograms per milliliter of Sambucus nigra lectin (SNA) (B- 1305–2; Vector Laboratories, Burlingame, CA, USA) was complexed with 1 µg streptavidin-HRP. For real-time PCR, RNA was extracted with the RNeasy Plus Tissue Mini Kit (Qiagen).