Alexa fluor 647 secondary antibody

Placental tissue sections were blocked with 4% BSA and 10% secondary antibody host serum in PBS/0.3% Triton X100 and incubated overnight with primary antibody solutions. Primary antibodies for cytokeratin 7 (1:500, Abgent, Cat#AJ1229a), CD163 (1:200, Thermo Fisher Scientific, Cat#MA1-82342), and Von Willebrand Factor (1:500, Dako, Cat#A0082) were used. To detect Cytokeratin 7 (CK7) and Von Willebrand Factor (VWF) goat anti-rabbit Alexa Fluor 647 secondary antibody was used (1:500, Cell Signaling Technology, Cat#4414) and displayed in white. CD163 primary antibody incubation was followed by goat anti- mouse Alexa Fluor 488 secondary antibody application (1:500, Invitrogen, Cat#A32723) and displayed in green. Sections were counterstained with DAPI, sealed with a coverslip using VECTASHIELD^® Antifade Mounting Medium with DAPI (Vector Laboratories, Cat# H-1200-10) and stored at 4°C until imaged. Representative images were captured on Nikon A1 confocal microscope (original magnification ×40) and prepared using FIJI software v.1.51h.

Flow cytometry analysis of cell-surface receptor CCR6 detection (using anti-CCR6, cat# 559562, BD Bioscience) was performed at 72 h as described earlier (15 (link)). To measure PKCα overexpression at protein level, FACS samples were collected six hours after in vitro transcribed PKCa RNA had been introduced to cells by nucleofection. The cells were fixed using pre-warmed BD Phosflow Fix buffer I (BD, Cat. no. 557870) for 10 minutes at 37°C. The cells were collected after centrifugation and resuspended in 300ul of cold Perm buffer III and incubated at +4°C for 30 minutes. Perm buffer was removed and cells were washed in 200ul of Stain buffer (BD Cat#554656). The PKCα antibody (Cell signaling Cat# 59754) was used in 1:100 dilution in staining buffer and the samples were incubated at room temperature for 30 minutes. Cells were washed two times and resuspend in staining buffer containing Alexa fluor-647 secondary antibody (cell signaling Cat no. 4414) for 30 minutes at room temperature. Cells were washed for two times and resuspend in PBS for FACS analysis. The data was acquired in BD LSR II/LSRFortessa. The acquired data were then analyzed either by Flowjo (FlowJo LLC) or Flowing software (https://bioscience.fi/services/cell-imaging/flowing-software/).