Cytation 1 device

Immunofluorescence staining was performed on tumor tissues. First, tumor tissue fixation was performed using 4% paraformaldehyde (Sigma-Aldrich, #HT5011). Next, heat-induced epitope retrieval was performed by incubating the tissue in 10 mM sodium citrate for 10 min. Subsequently, permeabilization was achieved by treating the tissue with 0.2% Triton X-100 (Sigma-Aldrich, #X100) for 5 min. To block nonspecific binding, the tissue was incubated in 5% skim milk for 40 min. Primary antibodies, including FOXP3 (eBioscience, San Diego, CA, USA, #11-5773-82), CD8 (eBioscience #53-0081-82), CD31 (Abcam, Cambridge, UK, #ab28364), F480 (Abcam #ab6640), CD163 (Abcam #ab182422), and CD206 (Abcam #ab64693), were used. The secondary antibodies used were FITC-conjugated AffiniPure F(ab’)2 fragment goat anti-rat IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA, #112-096-003) and Cy3-conjugated AffiniPure F(ab’)2 fragment goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch #111-166-003). The stained tissue was mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) #14-5773-82). Fluorescent images of the samples were captured using the Agilent BioTek Cytation 1 device. The images were analyzed using the Gen5 program (Version3, BioTek, Winooski, VT, USA) to extract relevant data and perform quantitative analysis.

After the incubation with SAG, PC3 cells were fixed using 4% paraformaldehyde (Sigma-Aldrich, Seoul, Republic of Korea). This was followed by the permeabilization of the cells using 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min. The fixed and permeabilized cells were then incubated in a blocking solution containing 5% skim milk for 1 h. PC3 cells were immunostained with primary anti-Ki67 antibody from Santa Cruz (Dallas, TX, USA) and secondary goat anti-mouse antibody from Invitrogen (USA). Mounting of the stained cells was performed with ProLong Gold Antifade Mountant with DAPI (Invitrogen; Waltham, MA, USA). Using the Agilent BioTek Cytation 1 device (USA), fluorescent images of the samples were acquired. The Gen5 program was used to extract relevant data from the images and conduct quantitative analysis.