Las af lite

The wing imaginal discs and pupal retinas were dissected from late third instar larvae and twenty-four hours after puparium formation (APF) pupae, respectively, then fixed for 17 min in phosphate-buffered saline (PBS) with 4% paraformaldehyde and then blocked in PBS with 0.3% Triton X and 5% BSA (bovine serum albumin) for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. After washed with PBS-Triton X three times, the wing discs were incubated with secondary antibody for 1 h at room temperature. The stained wing discs were then mounted on the slides with glycerol. Primary antibodies: mouse anti-Cut (2B10, 1:200, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), mouse anti-Delta (C594.B9, 1:200, DSHB), mouse anti-Notch (C17.9C6, 1:100, DSHB), mouse anti-β-gal (1:1000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-cleaved caspase-3 (1:200; Abcam). Secondary antibodies [anti-mouse Cy3 (1:1000), anti- rabbit Cy5 (1:1000)] were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The fluorescent images were acquired by confocal microscope TCS SP5 (Imaging Core, First Core Labs, National Taiwan University College of Medicine) and Axio Imager A1 Microscope (Zeiss, Thornwood, NY, USA). Leica LAS AF Lite and Adobe Photoshop were used to analyze and process images.

Dendritic cells were seeded on poly-lysine-coated glass coverslips for 12 h and then stimulated with zymosan particles. Cells were fixed with 10% formaldehyde in PBS and stained with anti-HIF1α Ab or anti-PKM2 Ab and goat anti-rabbit IgG Ab labeled with Alexa-Fluor^®480. The coverslips were observed by laser-scanning confocal fluorescence microscopy using a Leica TCS SP5 apparatus equipped with a white-light laser and a Leica 63PL APO NA 1.40 oil immersion objective. Image analysis and subcellular colocalization fluorograms were generated and analyzed using the Leica confocal software package LAS AF Lite and Adobe Photoshop CS5.1 software.