Orb4941

THP-1 macrophages were cultured in 24-well plates on tissue culture plastic. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed 3x in phosphate buffered saline containing 0.1% Tween-20 to permeabilize cells. Cells were then blocked with 5% BSA for 30 min in PBS. Primary antibodies for CCR7 (Novus Biologicals, NB100-712) or CD206 (Biorbyt, orb4941) were incubated at 1:100 in PBS with 1% BSA and 0.05% Tween-20 at 4°C overnight. Secondary antibodies (AbCam, ab150129, ab96883) were incubated at room temperature for 2 hrs. Images were taken with a Zeiss Axiovert 40 CFL.

Tissue construct samples (TTCs and CTCs) were fixed in 4% paraformaldehyde and then paraffinized to prepare tissue sections. Tissue sections were then stained histologically with hematoxylin and eosin (Thermo Fisher Scientific, USA), Masson’s trichrome (ScyTek Lab, USA), picrosirius red (ScyTek Lab, USA), Safranin O (ScienCell Research Lab, USA) and alizarin red (Sigma Aldrich, USA) separately. Tissue sections were also stained immunohistochemically with elastin (ab21610, abcam, USA), vimentin (ab92547, abcam, USA), α-smooth muscle actin (SMA) (ab124964, abcam, USA), INOS (ab3523, abcam, USA) and CD206 (orb4941, biorbyt, USA) antibody markers separately. For staining, protocols described in detail in our previously published papers were applied (Jana and Lerman, 2019 (link), Jana, Lerman and Simari, 2015 (link)).