Tcs sp2 uv confocal microscope

Fixed PfPanK2-GFP-expressing 3D7 strain P. falciparum parasites within infected erythrocytes were observed and imaged with a Leica TCS-SP2-UV confocal microscope (Leica Microsystems) using a 63× water immersion lens as described in the S1 Text. To confirm the expression of SaPanK-Ty1 in the TgPanK1-mAIDHA^+SaPanK-Ty1 line, immunofluorescence assays were performed based on the protocol described by van Dooren et al. [73 (link)]. T. gondii parasites were incubated with mouse anti-Ty1 antibodies (1:200 dilution). Secondary antibodies used were goat anti-mouse AlexaFluor 488 at a 1:250 dilution. The nucleus was stained with DAPI. Immunofluorescence images were acquired on a DeltaVision Elite system (GE Healthcare) using an inverted Olympus IX71 microscope with a 100× UPlanSApo oil immersion lens (Olympus) paired with a Photometrics CoolSNAP HQ² camera. Images taken on the DeltaVision setup were deconvolved using SoftWoRx Suite 2.0 software. Images were adjusted linearly for contrast and brightness.

Erythrocytes infected with trophozoite-stage 3D7 strain P. falciparum parasites expressing PfPanK1-GFP were observed and imaged either with a Leica TCS-SP2-UV confocal microscope (Leica Microsystems) using a 63 × water immersion lens or a Leica TCS-SP5-UV confocal microscope (Leica Microsystems) using a 63 × oil immersion lens. The parasites were imaged as fixed or live cells as described in the SI.

Lung tissue sections (5 μm) were deparaffinized in xylene, rehydrated in graded alcohol, and antigen retrieval was performed in EDTA (1 mM, pH 8.0) for 10 min in a microwave. Samples were blocked in 1% bovine serum albumin (BSA) and incubated overnight at 4°C with anti‐IL‐1α (AF‐200‐NA, R&D) or an IgG1 control antibody (both 10 μg/mL, 1% BSA). Slides were washed and incubated with a fluorescein isothiocyanate–conjugated anti‐goat secondary antibody (F7367, Sigma) for 2 h at room temperature. Slides were washed and mounted in VectorShield mounting media containing DAPI (Vector Laboratories, Burlingame, CA). Images were acquired using a Leica TCS SP2 UV confocal microscope.