Oregon Green 488 carboxylic acid

For analysis of fixed samples mounted in PPDM (90% glycerol, 0.5% p-phenylenediamine, 20 mM Tris-HCl pH 8.8), images were acquired on a DeltaVision deconvolution Olympus IX70 microscope (Applied Precision) equipped with a CoolSnap CCD camera (Roper Scientific) at 20°C using a 100×, 1.35 NA Olympus U-Planapo oil objective lens. Immunofluorescence of fixed embryos was performed as described (Desai et al., 2003 (link)), using the following rabbit antibodies at a concentration of 1 μg/ml: α–CAR-1 (Cy3-labeled; described above); α–AIR-2 (Cy-5 labeled; generated against a GST fusion to the full-length protein); α–ZEN-4 (Cy-5 labeled; generated against a GST fusion to the COOH-terminal 108 aa); the mouse monoclonal antibody DM1α (Oregon green 488–labeled; Sigma-Aldrich); the goat polyclonal GFP antibody (Oregon green 488–labeled; generated against a 6x-histidine fusion to the full- length protein); and the unlabeled rat CGH-1 antibody (JDCR5; a gift of K. Blackwell, Joslin Diabetes Center, Boston, MA). For analysis of gonads, the tails of adult hermaphrodites were amputated in 5% sucrose and 100 mM NaCl to extrude the gonads. Fixation and immunofluorescence on gonads was performed as described for embryos. For live analysis, embryos were mounted on agarose pads as described previously (Oegema et al., 2001 (link)), and imaged on a spinning disc confocal microscope (Nikon Eclipse TE2000-E) equipped with a Hamamatsu Orca-ER CCD camera at 20°C using a Nikon 60×, 1.4 NA Planapo oil objective lens. For osmotically sensitive embryos and embryos imaged in the absence of compression, filming was performed in a depression slide containing meiosis media (25 mM Hepes at pH 7.4, 60% Leibowitz L-15 Media, 20% FBS, 500 μg/ml inulin) and sealed with petroleum jelly. Analysis of spindle pole separation, spindle microtubule density, and furrow movement was performed using Metamorph software.
Kymographs were constructed by compressing the image of the furrow region from each time point (same region as in Fig. 5 B) to a single vertical line, in which the maximum intensity along the x-axis of each original image is displayed for each point along the y-axis. The vertical strips for sequential time points are laid adjacent to each other so that time increases from left to right along the x-axis.

Glass coverslips were prepared essentially as described previously (Mueller et al., 1992 (link)). In brief, they were coated with Oregon green^® 488-conjugated gelatin (0.2 mg/ml in 2% sucrose buffer), cross-linked 15 min in 0.5% glutaraldehyde in PBS, and incubated for 3 min with 5 mg/ ml NaBH₄ in PBS. After quenching with DME at 37°C, cells were plated on coated coverslips in DME containing 10% FCS with or without inhibitors and incubated at 37°C for 3 or 4 h before processing for immunostaining. Results were quantified by counting cells degrading matrix, as defined by ability to form at least one degradation patch regardless of its size, and represented as a percentage of the total (50 cells per treatment in at least two independent experiments).

A Zeiss LSM 710 confocal microscope (Carl Zeiss) was used to acquire spectral images. All images were acquired with Plan-Apochromat 20x 0.8 NA objective in lambda mode with 29.1 nm channel bandwidth resulting in 9 channels detected over the visible spectrum. Simultaneous excitation with 405 nm, 488 nm and 561 nm lasers was used. 3-D z-stack images were acquired as tile scans with 9 z-planes centered in the middle of the gut spanning 8.3 μm total in z dimension. Linear unmixing was performed on the spectrally acquired images after stitching the tiles together in ZEN software v 3.4. We extracted the reference spectra for DAPI, EUB 338 Alexa flour 594, WGA Oregon green 488 and autofluorescence from the labelled zebrafish and applied them for the linear unmixing.