Ab106350

THP-1 cells (1 × 10⁶ cells) were washed in PBS and coated on slides using cytospin technique at 600 rpm for 3 min. The slides were then fixed in 4% formaldehyde for 10 min and washed three times in cold PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed three times in cold PBS, blocked in 1% bovine serum albumin (BSA) for 1hr, and then incubated overnight with primary antibody (1:100 dilution, rabbit anti-human IL-8 polyclonal antibody; Abcam^® ab106350) at room temperature. Cells were washed three times in PBS/Tris buffer and incubated with secondary antibody (goat anti-rabbit antibody conjugated with Alexa Fluor^® 488; Abcam^® ab150077) for 1hr. After several washes in PBS, the cells nuclei were counterstained with 4′,6-diamidino- 2-phenylindole (DAPI) and mounted (Vectashield, Vectorlab, H1500). Confocal images were collected using inverted Zeiss LSM710 Spectral Confocal Microscope (Carl Zeiss, Gottingen, Germany) and EC Plan-Neofluar 40 ×/1.30 oil DIC M27 objective lens (Carl Zeiss, Gottingen, Germany). After sample excitation using a 405 nm and 488 nm line of an argon ion laser, optimized emission detection bandwidths were configured using Zeiss Zen 2010 software (https://www.zeiss.com/microscopy/int/products/microscope-software.html).

We obtained surgical specimens from 30 patients with primary tongue OSCC who were treated in the Department of Oral and Maxillofacial Surgery at Kyushu University Hospital from 2005 to 2018. The sections were deparaffinized in xylene and then hydrated by graded series of ethanol. The sections were then incubated with the following primary antibodies at room temperature: mouse anti-CD163 (Clone 10D6, 1:400 dilution; Novocastra, Newcastle, UK), rabbit anti-CD204 (ab217843, 1:200 dilution; Abcam, Tokyo, Japan), rabbit anti-CD206 (ab64693, 1:1000 dilution; Abcam), rabbit anti-IL-8 (ab106350, 1:500 dilution; Abcam), and rabbit anti-PAI-1 (ab66705, 1:1000 dilution; Abcam). Samples were washed with TBST, and 100–400 μL of DAB (Peroxidase Stain DAB Kit; Nacalai Tesque, Kyoto, Japan) was applied as a chromogen to each section. Finally, Mayer’s hemalum solution (1:4 dilution; Merck KGaA, Darmstadt, Germany) was used for counterstaining, and then sections were washed twice for 5 min each in dH₂O. After dehydration, sections were mounted with coverslips.