11 June, 2009

Actin Belly

40X coronal stomach section, stained for actin. Click for high res. Scale bar = 200um.

Frozen section, wash off excess OCT in RT dH2O, 10min permeabilization in 100% acetone, 30min block in Toaster's Übermegablocking Buffer*, added primary polyclonal Ab without washing, incubate 1h @ RT, wash 3X in Washy Wash Buffer**, add labeled actin at 1:200 and secondary labeled Ab against primary at 1:10, incubate 1h @ RT, wash 3X in Washy Wash Buffer, tap off all excess wet, 2 drops Prolong Gold Antifade + DAPI (@RT), coverslip w/out bubbles, cure O/N in a dark box.

The resolution I managed to get here is so exquisite and the result so beautiful that I am very tempted to get a 5'x6' giclee print of it and hang it on my everything.

*Toaster's Megablocking Buffer: 1% BSA, 1% FBS, 1% NGS in 1X PBS; to make Toaster's Übermegablocking Buffer add 1:10 CD16/CD32 to block endogenous Fc antibody receptors in the tissue.
**Washy Wash Buffer: 2% FBS, 0.02% NaN2 in 1X PBS


Juniper Shoemaker said...

It's just like a red jewel. It's lovely!

Ambivalent Academic said...

Dude, why do your scale bars always run off the left side of the image?

Ha! Verification word = "scalized"

Hermitage said...

Gotta agree with AA here. Am I supposed to actually interpret that crazy skinny white line as a scale bar? Come on now, archnemesis!

Toaster Sunshine said...

The program in the microscope core facility that adds in the scale bar is incredibly inflexible with regards to the scale bar's size or its placement. I'm pretty much limited to changing the color, thickness of the line, and font of the numbers on top, which in my opinion take up far too much space.

And I said it's 200um in the caption, so don't go telling me that it isn't a scale bar. I know a jerboa from a hamster.

Ambivalent Academic said...

Yeah but it runs off the side of your image. How do I know where the other end of 200um is if I can't even see it? Am I supposed to guess? Come on, you can't publish that shit if you have your scale bars sliding off into nothingness.

Our acquisition software makes shitty scale bars too, so I save 2 images - one with and one without. Then I go in and add a better looking one to the image without a bar with Illustrator or Photoshop that is based on the software's bar.