Alexa fluor 555 labeled secondary antibody

Immunofluorescene immunohistochemistry (IF-IHC), slide scanning, and quantitative analysis of digitized images were performed in a manner blinded to outcome. Fluorescence immunohistochemistry for CD163 was performed on an autostainer (Agilent/Dako Omnis) using anti-CD163 antibody (Cat# HPA046404, 1:4000; Sigma) followed by HRP-conjugated secondary antibody (Cat# K4003, Agilent) and visualized using Cy5-tyramide as substrate (Cat# NEL745001KT, Perkin Elmer), multiplexed with anti-pan-cytokeratin antibody (mouse monoclonal AE1/AE3 blend, Cat# M3515, 1:100, Agilent) with Alexa-Fluor-555-labeled secondary antibody (Cat# A21422, Thermo Fisher) to identify cancer cells, followed by counterstaining with DAPI to visualize cell nuclei as previously described [42 (link),43 (link),44 (link)]. Stained slides were scanned at 20× magnification on the Pannoramic Flash 250 scanner (3DHistech) and fluorescent images were captured in three channels (Cy5, Cy3/Alexa-555 and DAPI). Cell segmentation of digital images was performed by TissueStudio software (Definiens) and CD163 immunoreactivity was computed for individual cancer cells and stromal cells for each tumor core. The operators were blinded to patient IDs and clinical outcomes. Some tissue cores were excluded from further analysis if the quality of the staining was deemed substandard or if less than 10 cancer cells were identified.

The five OC cell lines expressing LIMD2 were processed for immunofluorescence based on similar conditions to the ones described above [14 (link)]. The cultured cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were permeabilized with 0.01% Triton X-100 and blocked with 3% BSA for 1 h at room temperature. The primary antibody against LIMD2 (catalog number: 15,471-1-AP; Proteintech, China) was incubated overnight at 4°C and Alexa Fluor 555-labeled secondary antibody (A-21428, Thermo Fisher Scientific, USA) was incubated for 1 h at room temperature. Slides were mounted with DAPI (Sigma-Aldrich, Germany) and visualized using an Olympus microscope (Olympus, Japan).

Cells were grown on eight-well chamber slides. Briefly, after being treated as indicated, the cells were fixed with 4% paraformaldehyde for 15 min. Cells were then washed in phosphate-buffer saline (PBS) followed by incubation for 2 h at 4 °C with rabbit anti-ZO2 (1:200, Invitrogen), anti-Occludin (1:200, Abcam, Cambridge, CA, USA), and anti-VE-cadherin (1:200; Abcam) antibodies. The slides were incubated at room temperature with Alexa Fluor 555-labeled secondary antibody (1:500, Invitrogen) for 1 h. The slides were analyzed with sequentially scanning on an Olympus laser scanning confocal microscope (Fluoview FV1000, Olympus, Japan). Image Pro Plus software was used for the analyses of co-localization and Pearson’s correlation coefficient [35 (link)] was used to represent the co-localization coefficient.