4 colors!   2 comments

Here’s a 4-color image of the cells I fixed and stained yesterday. The cells were in differentiation conditions for 1 week before fixation:

Hopefully the color balance will translate ok- being color blind makes this stuff more difficult.

Here’s the color key:
Blue: DAPI nuclear stain
Green: SSTR3:GFP chimera
Red: SSTR3 stain
White: Acetylated alpha-tubulin

It’s a little different than I wanted: I didn’t have a red stain for PC2 (my PC2 primary is anti-rabbit, I don’t have a texas red: anti-rabbit secondary). Some observations:

1) According to the SSTR3 stain, the somatostatin receptor is primarily localized to the nucleus in ciliated cells. In cells without a cilium, the SSTR3 stain co-localizes well (the yellow spots) with the SSTR3:GFP chimera (which indicates that the chimera is indeed a chimera, and that the GFP hasn’t split off, the SSTR3 is recognizable and not degraded, etc.), but is *not* located in the nucleus. This is interesting: under what comditions does SSTR3 go to the nucleus? Does this mean the chimera is cleaved? The hope (was) that the chimera could be used to perform live-cell fluorescence imaging of the cilium to increase its visibility… doesn’t look promising.

2) my primary and secondary antibodies work, specifically target the proteins they are supposed to, and I know how much antibody to add (about 1 microgram).

3) the 4-color cube works, there is no ‘bleeding’ between color channels.

Today I’m giving a seminar to celebrate the movie “Powers of 10”, by Ray and Charles Eames. I first saw the film when I was in elementary school, and it definitely stoked my interest in science.


Posted October 6, 2010 by resnicklab in Physics, Physiology, Science

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2 responses to “4 colors!

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