Goat anti mouse igg h l alexa fluor 647

For staining of cell surface antigen, cells were detached with Cellstripper (Corning) and resuspended in PBS with 0.5% FBS and 1 mM EDTA (FACS [fluorescence-activated cell sorting] buffer). Bst2 was stained with phycoerythrin (PE)-labeled anti-Bst2 (BioLegend, clone 129C1; 1:500 in FACS buffer) and anti-mouse CD16/CD32 (eBioscience; 1:250 in FACS buffer) on ice for 45 min. Ifnar1 was stained with 1 μg/ml of anti-mouse Ifnar1 (clone MAR1-5A3; BioXcell) at room temperature for 45 min, followed by incubation with a goat anti-mouse IgG (H+L) Alexa Fluor 647 (Thermo Fisher, catalog no. A21236; 1:1,000 in FACS buffer) at room temperature for 30 min. For cells infected with GFP-labeled viruses (SINV-EEEV-GFP and VSV-GFP), cells were detached with trypsin and fixed with 1% paraformaldehyde (PFA) at room temperature for 15 min. Flow cytometry was performed on a MACSQuant Analyzer 10 (Miltenyi Biotec), and data were analyzed using FlowJo 10.6.1.

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.

A total of 4 × 10⁵ cells were seeded on coverslips, which were placed in a 12-well plate for 24 h. The cells were treated with 20 nM paclitaxel or DMSO (control) for 24 h and then fixed in 4% paraformaldehyde PBS (Wako Pure Chemicals, Tokyo, Japan) for 20 min at 37 °C. After blocking with PBS containing 0.1% saponin (MP Biomedicals, Santa Ana, CA, USA) and 3% bovine serum albumin (BSA) for 30 min, the cells were incubated with primary antibodies for 1 h, followed by incubation with secondary donkey anti-rabbit IgG H&L, Alexa Fluor 405 (Abcam, Cambridge, UK, ab175651) or goat anti-mouse IgG (H+L), Alexa Fluor 647 (Thermo Fisher Scientific, Boston, MA, USA)-conjugated antibodies (diluted in PBS containing 0.1% saponin, 1:500) for 1 h; all reactions were carried out at room temperature. Coverslips were picked and mounted with DAPI-Fluormount-G^® (SouthernBiotech, Birmingham, AL, USA). Finally, the cells were observed under a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). The dilutions used for the primary antibodies were as follows: GFAP (1:300; Bioss, Woburn, MA, USA, bs 0199R) and Ki67 (1:200) (Proteintech, Rosemont, IL, USA, 27309-1-AP). All primary antibodies were diluted in PBS containing 0.1% saponin and 3% BSA. The obtained images were analyzed using the ImageJ2 software (National Institute of Health, Bethesda, MD, USA).

The cells were inoculated on slides and grown to approximately 70% confluence. The slides were washed twice with phosphate-buffered saline (PBS) (Gibco, Waltham, MA, USA). The slides were fixed with 4% paraformaldehyde for 20 min, blocked with 3% BSA for 30 min, and incubated with primary antibodies against p-AMPK, collagen I, IL-8 (Cell Signaling Technology, Boston, MA, USA), vimentin, desmin, keratin, and S100 calcium binding solution (Thermo Fisher Scientific, Rockford, IL, USA) overnight at 4 °C. The slides were washed again and incubated with CY3-labeled goat anti-mouse secondary antibodies (Servicebio, Wuhan, China), goat anti-mouse IgG H&L (Alexa Fluor 647), and goat anti-rabbit IgG H&L (Alexa Fluor 488) (Thermo Fisher Scientific, Rockford, IL, USA) at room temperature in the dark for 1 h. Nuclei were stained for 5 min with DAPI (Servicebio, Wuhan, China). After a final wash, an anti-fluorescence quenching agent (Servicebio, Wuhan, China) was added to mount the slides, and images were collected with a microscope (Nikon, Tokyo, Japan).

Casper3-GR (FP971; Evrogen) vector was digested with BamH1 and Not1 to remove TagRFP-linker-TagGFP. The same digestion was performed for pMAX vector and both vector and insert were ligated. The following primary antibodies were used: anti-GAPDH (RRID:AB_561053), anti-tRFP (RRID:AB_2571743) and anti-GzmB (RRID:AB_2114432). Secondary antibodies were HRP-conjugated donkey anti-rabbit and Alexa Fluor 647 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific). For structured illumination microscopy (SIM), Alexa647-coupled anti-granzymeB (RRID:AB_2294995) and Alexa647-coupled anti-perforin (RRID:AB_493255) antibodies were used. For total internal reflection fluorescence microscopy (TIRFM) a hamster anti-mouse CD3ε (RRID:AB_394591) antibody was used for coating coverslips and stimulating cells. For FACS, a rat anti-mouse CD107a-PE (LAMP-1-PE) (RRID:AB_1732051) antibody was used.

Tissues were deparaffinized and rehydrated as described previously (De Nisco et al, 2019 (link)). Following antigen retrieval in 10 mM citrate buffer, tissues were blocked in 5% goat serum (Krenacs et al, 2010 (link)). IF was performed with primary antibodies against COX-2 (D5H5) 1:500 (rabbit; Cell Signaling) and 2 μg/ml neutrophil elastase (ELA-2, 950334, mouse; Novus). Secondary antibodies Alexa Fluor-555 goat anti-rabbit IgG(H+L) and Alexa Fluor-647 goat anti-mouse IgG(H+L) (Thermo Fisher Scientific) were added to a final concentration of 4 and 2 μg/ml, respectively. Hoechst 33342 final concentration was 1 μg/ml. Alexa Fluor-488 phalloidin and Alexa Fluor-488 Wheat Germ Agglutinin were used at a 1:500 dilution.