help chosing a fluorescent DNA stain - (Nov/28/2006 )

We work on FIXED hamster spinal cord tissue that is cryosectioned (14um). I want to do an IHC experiment using three fluorescent labels and image the material on a confocal microscope. I am looking for a third fluoresecent probe to run with Alexa 488 and Cy3 or Texas Red. I can use either a far red probe such as To-Pro-3 or Draq5 or one that excites around 400nm such as Hoechst or DAPI. Invitrogen offers a ProLong Gold antifade media with the DAPI already dissolved so its a one step application which would be optimal if anyone can tell me it works on fixed tissue sections and not just cells. I am having a hard time finding any info. If anyone has used paraformaldehyde fixed tissue sections with any of these probes I would really appreciate any feedback +/- that you can provide. Thanks!

I say go with Hoechst. I triple labeled paraformaldehyde fixed, cryosectioned rat retinas with Alexa 488 (also Oregon Green), Cy3 and Hoechst and visualized by confocal and it worked beautifully. Although the DAPI in the mounting media is convenient I find you get a lot of diffuse background blue staining. Hoechst gives a much cleaner more crisp stain than the DAPI in the mounting media. DAPI alone (not in the mounting media) followed by rinsing the sections would probably give a similar result to Hoechst but I never tried it. We had Hoechst on hand so I went with that.

Info I find on Hoechst is usually with xylene and alcohol washes before a 5 minute stain in Hoechst. Can you use Hoechst without these? We typically do washes with PBS/Triton-X and then into goat serum before primary antibody application. The next day we repeat, add the secondary antibody, then wash with PBS before coverslipping. Can I do the Hoechst stain after the secondary antibody and a couple of washes?

Sounds like you do your immunos similar to me. I just checked my lab book and for the Hoechst staining I would add it following the secondary antibody and after the washes as follows:

Hoechst staining – added a 1:100 dilution (diluted in PBS) of Hoechst (10mg/ml) to the appropriate slides. Incubated at room temp for 5 minutes with gentle agitation. Washed 3X with PBS. Then mounted in Vectashield mounting media.

So no I didn't do any xylene treatments or alcohol washes.

I used Hoecsht 33342 from Invitrogen (cat # H3570) that was already in solution. I used it quite concentrated (1:100 dilution of the stock) because our confocal was testy about picking up signal in the blue spectrum so I needed a lot of staining.