Mab1430

Mouse brains were formalin-fixed and paraffin-embedded, and 4-µm sections were cut for hematoxylin and eosin (H&E) staining. For immunofluorescence, sequential staining was used. First, GBM tissue sections were stained with mouse anti-FGL2 antibody (1∶100, H00010875; Novus Biologicals, Littleton, CO, USA) overnight at 4 °C, followed by 1 h incubation with anti-mouse Alexa Fluor 647-conjugated antibody. The sections were blocked with Fab fragment anti-mouse IgG (15-007-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h, and then stained with rabbit anti-CD31 antibody (bs-0468R, 1:100; Bioss Inc., Woburn, MA, USA), mouse anti-CD45 antibody (MAB1430, 10 µg/mL; R&D Systems), or Alexa Fluor 488-conjugated GFAP (53-9892, 5 μg/mL; Thermo Fisher Scientific) overnight at 4 °C, followed by incubation with Alexa Fluor 488-conjugated anti-mouse/rabbit antibody (1∶300; Invitrogen) for 1 h. ProLong Gold Antifade Mountant with 4′-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was used as the mounting medium. Slides were further processed for imaging analysis using an Olympus Fluoview FV1000 microscope.

After fixation, cultures in the OrganoPlate were stained for immunofluorescent markers. As described previously, in short, cells were permeabilized using a Triton X-100 solution for 10 min and blocked using a buffer containing FBS, bovine serum albumin, and Tween-20 for 45 min (42 (link)). Primary antibody was incubated for 1–2 hours or overnight, after which secondary antibody was incubated for 1 hour. The following primary antibodies were used to stain fixed cultures: Anti‐VE-Cadherin 1:500 (Abcam, ab33168), anti‐ICAM‐1 1:50 (R&D systems, BBA3), anti-human CD45 (R&D systems, MAB1430). The following secondary antibodies were used to stain fixed cultures: Goat anti‐rabbit IgG (H+L) Alexa Fluor 488 1:250 (Thermo Fischer Scientific, A11008), Goat anti‐mouse IgG (H+L) Alexa Fluor 647 1:250 (Thermo Fischer Scientific, A21428) and CF647 Goat anti‐mouse IgG (H+L) Alexa Fluor 647 1:250 (Biotium, 20040). Nuclei were stained using Hoechst (ThermoFisher, H3570). After staining, the OrganoPlate was transferred to a confocal high content imaging microscope for automated imaging (Micro XLS-C, Molecular Devices). Images were acquired at 10x magnification at 3 µm increments along the height of the microfluidic channel. Analysis was based on Sum Projection (ICAM expression) or Max projection (VE cadherin) images of the top and bottom 10 z-slices.

Images were acquired using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany) with a 40× objective/1.4 numerical aperture oil PlanApochromat immersion lens. Alexa-fluor 488 fluorescence was detected using the 488 nm laser line of an Ar laser and an LP 505 filter. TRITC fluorescence was detected using a 561 nm HeNe laser (1 mW) and an LP 560 filter and Alexa-fluor 633 fluorescence was detected using a 633 nm HeNe. Z-stacks were acquired to confirm the intracellular localization of TRITC labelled AuNRs. The thickness of the slices and the interval between slices were set to 0.7 µm. After irradiation, cells were deposited onto a glass slide using cytospin centrifugation (800 rpm, 5 min), fixed with 4% PFA for 20 min and stained for confocal microscopy. Cell membrane was stained with monoclonal mouse anti-human CD45 antibody, diluted 1:50 (R&D Systems, MAB1430) and using Alexa Fluor-488 goat anti-mouse igG as secondary antibody, diluted 1:1000 (Life technologies, A11001). For staining early endosomes, cells were permeabilized with 0.1% TRITON X-100 for 10 min and stained with rabbit anti-EEA1 monoclonal antibody (Cell Signaling Technology, 3288), diluted 1:100. Alexa Fluor-633 goat anti-rabbit (Life Technologies, A21070), diluted 1:1000 was used as secondary antibody. Cell nuclei were stained with DAPI.