Sunday, August 5, 2012

Chris - Week 5: Anti-Tubulin Confocal Microscopy

During Week 5 I started a new AB staining procedure, this time targeting tubulin. This protein makes up the cilia themselves, unlike centrin which was merely a component of the basal bodies. For my purpose, the anti-tubulin antibody would seem to be superior to the anti-centrin because the cilia are clearly labeled, eliminating the guesswork that came with labeling centrin. Note that in the following confocal micrographs, the white channel represents nuclei stained with Hoechst, while all the green structures are stained with anti-tubulin.

Figure 1: An  anterior/posterior view of a 10 hour
old M. alaskensis larva

The ten hour old M. alaskensis larva in Figure 1 has a patchy distribution of short cilia. There  appears to be a pattern, with 4 groups of multi-ciliated cells arranged along the equatorial region of the specimen. This pattern was seen to hold for all the 10 hour specimens examined.
Figure 2: An  anterior/posterior view of a 12.5 hour
old M. alaskensis larva

 The twelve and a half hour old M. alaskensis specimen in Figure 2 is relatively well ciliated along the equatorial region. In addition, cilia are clearly beginning to develop across the anterior/posterior surfaces.

Figure 3: A  posterior view of a 15 hour
old M. alaskensis larva

The 15 hour specimen in Figure 3 possesses well developed cilia along the equatorial region, noticeably more than in Figure 2. It is not clear from this Z-projection, but this larva is also ciliated on what Svetlana predicts is the anterior portion. Thus, the bald surface shown is likely the posterior of the specimen. 

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