Eclipse te2000 u inverted compound fluorescence microscope

Rates of neutrophil apoptosis were assessed by dual staining with Rhodamine-TUNEL and FITC-TSA. Transgenic zebrafish larvae were injured at 8 dpf and fixed at 24 hpi in 4% paraformaldehyde. TUNEL (ApopTag Red; Millipore Corp.) staining labeled apoptotic cells with red fluorescence, and TSA (TSAplus kit; Fluorescence Systems, PerkinElmer Life and Analytical Sciences) staining labeled neutrophils with fluorescein green fluorescence. Neutrophils at the wound were imaged on an Eclipse TE2000-U inverted compound fluorescence microscope (Nikon), and neutrophil-specific apoptosis was assessed by the percentage of TSA-positive neutrophils labeled with TUNEL. Red TUNEL–positive–only cells were assessed for total apoptosis rates.

To assess neutrophil migration behavior before and after interaction with a macrophage, transgenic larvae Tg(mpx:EGFP)i114 were crossed to Tg(mpeg1:mCherry-CAAX)sh378. Three days post fertilization larvae were injured, and images were taken every 3 min between 0 and 12 hpi using an Eclipse TE2000-U inverted compound fluorescence microscope (Nikon). Direct contact was defined as when cell membranes could be seen to touch. Comparisons of migration behavior were made over a 30-min period before and after interaction.