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For an even bleach line, reload your brush every two inches.
Less than 20% recovery was observed after bleaching cell lines stably expressing YFP-Bub1 [10], and in cells transiently transfected with GFP-Bub1 56% recovered with a t1/2 of ∼30 seconds [8].
And of course the various washes, and the intensity of bleach-line "whiskering" and the degree of destruction in the "destructed" styles.
Embryos were collected for 5 hours at 21°C on apple juice agar plates, aged for 17 hours at 18°C, dechorionated in 50% bleach, lined up on slides using heptane glue, dehydrated for 14 minutes over silica gel and covered with Halocarbon oil (Halocarbon, New Jersey, U.S .. Microinjection was performed at stage 13 following standard techniques.
Images shown were taken at t = 0 (top panel) and right after bleaching (middle panel); the red line defines the bleached region.
The Golgi area (within the dotted line) was bleached, and the time course of entry of PC-III-GFP to the TGN was monitored.
(D ) Recovery curves along X -axis (dots) and fit of the diffusion model to the post-bleach (red line).
One hundred percent of a 561 nm laser line was used for bleaching a small rectangular region of interest in a single scan.
(C ) Normalized intensity profile along the X -axis at the first time point after bleaching (dots) and Gaussian fit (red line).
Time-lapse mode was used to collect one prebleach image for each channel followed by bleaching with 50 iterations of the 514 nm argon laser line at maximum power (to bleach YFP).
Time course: 20 line scans for pre-bleaching (0.8 s), 5 for bleaching (0.2 s), and 500 for post bleaching (20 s).
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