Dm2000 fluorescence microscope

Fluorescein isothiocyanate (FITC) labeling of mature normal RBCs (nRBCs) was performed as described previously.^{19 (link)} The FITC-labeled nRBCs (FITC-nRBCs) were cultured in P. falciparum complete culture medium before they were used in FITC-Dextran transfer assays. For light and fluorescence microscopy, parasite-infected RBCs were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) in PBS and were examined at the indicated time points using a Leica DM2000 fluorescence microscope (Leica, Bensheim, Germany). For indirect immunofluorescence assay, thin blood smears were incubated with a mixture of anti-Ago2 mouse monoclonal antibody (ab 57113, Abcam, Cambriage, MA, USA) and anti-parasite cytoplasm rat monoclonal antibody 5H2C6V (maintained in our lab). Normal mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as a negative control. FITC-conjugated goat anti-mouse IgG and AlexaFluor 594-conjugated donkey anti-rat IgG were used as secondary antibodies. The slides were mounted with mounting medium containing DAPI (ZSGB-bio, Beijing, China) and were analyzed using a confocal Leica TCS-SP2 fluorescence microscope (Leica). The transfer potential of FITC-Dextran directly toward RBCs was evaluated with 5 × 10⁷ nRBCs cultured in medium containing 0.5 mg/mL FITC-Dextran for 24 or 72 h and then analyzed by flow cytometry (Supplementary Figure S1).

Fluorescent images were captured by using a Leica DM2000 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a CCD high resolution cooled camera, using the Leica Application Suite software (Leica Microsystems).
Immunocytochemical staining was evaluated by image analysis as previously described.
²³ (link) For PSR staining, a total of 10 fields per well were randomly chosen and images were viewed with brightfield illumination at 40×. Image analysis was performed using the Leica Q500 MC Image Analysis System (Leica, Cambridge, UK).

Invasion was measured using Matrigel-coated invasion inserts of 8μm pore (Corning), according to the manufacturer’s instructions. In the upper compartment, 2.5 × 10⁴ RKO cells were cultured at 20% O₂ or 1% O₂ conditions, either alone or stimulated with macrophages cultured at 20% O₂ or 1% O₂ at the lower compartment. Cells were allowed to invade for 24 h at 37 °C, at 20% O₂ or 1% O₂, respectively. After this period, the inserts were washed with PBS and fixed in 4% PFA for 20 min at RT, and the non-invading cells in the upper compartment of the inserts were removed. The filters were mounted in Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and the invading cells were visualized and counted with a Leica DM2000 fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

LLC-PK1 cells and dGBECs were washed twice with Ca²⁺/Mg²⁺ PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO_4, 1 mM MgCl₂ and 0.1 mM CaCl₂), fixed with 4% paraformaldehyde in PBS for 30 min at 25°C and then immediately permeabilized with 0.2% Triton X-100 in PBS for 10 min at 25°C. Nonspecific binding sites were blocked with 0.2% gelatin in PBS for 5 min, and the slides were then incubated with primary antibodies (anti-megT, 1:500; anti-E-cadherin, 1:500; or anti-pSMAD2/pSMAD3, 1:100) overnight at 4°C. The coverslips were then washed with blocking buffer and incubated with secondary antibodies (Alexa Fluor 488 Rabbit, Alexa Fluor 555 Mouse and Alexa Fluor 488 Mouse; all 1:500) for 2 h at 25°C. The coverslips were then washed with PBS and mounted on a slide with a drop of Fluoromount-G. Finally, the slides were examined using a Leica DM 2000 fluorescence microscope.

Auto-fluorescence images were acquired with a Leica DM2000 fluorescence microscope. The illumination source was a blue LED with a wavelength of about 470 nm. The color images were obtained for wavelengths above 500 nm using the attached Leica DFC450 C camera and the Leica application suite lite software. The same microscope and filter settings were used for the thioflavin-S-stained tissue sections after spontaneous Raman and SRS imaging.

TUNEL assay was carried out using the ‘‘In situ cell death detection kit—fluorescein’’ (Roche, Boulogne-Billancourt Cedex, France) as previously described [47 (link),48 (link)]. Briefly, cells were fixed in 4% paraformaldehyde (PFA) and cytospins were prepared. Cells were then permeabilized in ice-cold 0.1% Triton X-100 in 0.1% sodium citrate and incubated with TUNEL reaction mixture (enzyme dilution 1:20). Slides were mounted in Vectashield Mounting Media with DAPI (Vector Laboratories Inc., Burlingame, CA, USA), observed in a DM2000 fluorescence microscope (Leica, Wetzlar, Germany) and a semi-quantitative evaluation was performed by counting a minimum of 500 cells per slide. In addition, a specific assay for apoptosis was carried out using the “Human Annexin-V-FITC/PI apoptosis” kit (Bender MedSystems, Vienna, Austria) as previously described [49 (link)]. All flow cytometry analyses were performed using the FACSCalibur flow cytometer (BD Biosciences), plotting at least 10,000 events per sample and using the FlowJo 7.6.5 software (Tree Star, Inc.).