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Hello,

Hopefully this can get you started. I would use a 2.5% glutaraldehyde and 2% formaldehyde in 0.1M phosphate buffer with a pH of 7.2.

For fixation of cells, add fixative at 2x concentration 1:1 to the cell media. Leave it for 1 hour at RT. If the cells grow in suspension, spin them down in the fixative at 3000rpm for 3 minutes. Leave it for 1 hour at RT in the fixative.

For fixation of tissue, perfusion fixation is preferred. For this, we recommend using 2% formaldehyde and 2.5 glutaraldehyde in 0.1M phosphate buffer, pH of 7.2. After perfusion, dissect out the tissue of interest, immerse in fresh fixative and cut into 1-2mm cubes, then leave it to fix at least 2 hours at RT.

Bring your fixed samples to the EM lab in the fixative, and then wash in phosphate buffer 3x 1% osmium tetroxide (in H2O), then leave for 1 hour at RT.

I hope this helps! Please let us know if we can be of any further assistance.

-Electron Microscopy Sciences

Hello!

Caulfield's is somewhat outdated, and most labs do not rely on it anymore. Most people use a combination of osmium with glut and a phosphate buffer. Sodium barbital is going to be very hard for you to get anymore without licensing, so this may be a better option for you.

Good luck and let us know if there is anything else we can do to help!

-Electron Microscopy Sciences