Counterstain using RuO4

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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monirathhav
Posts: 15
Joined: Sat Feb 24, 2018 6:33 am

Counterstain using RuO4

Post by monirathhav » Fri Jul 06, 2018 6:17 pm

Hi,

Has anyone tried counterstaining with Ruthenium Tetroxide as suggested in this latest IMC paper? https://www.ncbi.nlm.nih.gov/pubmed/29405336

I was wondering if RuO4 step comes before or after the intercalator step. Or is intercalator not necessary at all here if we already include Histone3 as nuclear marker?

Thank you.

Monirath

mattbliss90
Posts: 9
Joined: Wed Jan 17, 2018 11:37 pm

Re: Counterstain using RuO4

Post by mattbliss90 » Fri Jul 06, 2018 7:02 pm

Monirath,
We tried it a few weeks back after the DNA intercalator stain. It worked well, but it is fairly strong so I wouldn't stain longer than outlined in the paper.

Matt

RaulCatena
Posts: 25
Joined: Mon Nov 27, 2017 12:49 pm

Re: Counterstain using RuO4

Post by RaulCatena » Fri Jul 06, 2018 9:45 pm

Hi monirathhav,

Like Matt says, right after the intercalator should work well. Remember to dilute the RuO4 in raw PBS and not in TBS. Also, make sure there is no serum either. RuO4 would react with the protein in the media (or with TBS too).

@Matt. If the signal is too strong just dilute a bit more. I would advise to do a titration at least every time you aliquot an ampulla of RuO4. Once you freeze the aliquots, and thaw one, you can do the titration and you will get consistent results with all the other aliquots, as once the RuO4 is frozen does not oxidize/lose strength anymore. Freezing in glass vials is optimal, by the way. The final ¨strength¨of your aliquots is going to be dependent on the time, temperature and conditions during the aliquoting procedure. It should be roughly around 0.0005% w/v final working concentration, but +/- 5x depending on those things.

Regards,

Raúl

monirathhav
Posts: 15
Joined: Sat Feb 24, 2018 6:33 am

Re: Counterstain using RuO4

Post by monirathhav » Tue Jul 10, 2018 6:11 pm

Hi Matt and Raul,

Thanks for the comments. Raul, do you mean diluting 0.5% RuO4 1000 times in PBS to get the 0.0005% RuO4 counterstain solution?

Monirath

RaulCatena
Posts: 25
Joined: Mon Nov 27, 2017 12:49 pm

Re: Counterstain using RuO4

Post by RaulCatena » Tue Jul 17, 2018 9:40 pm

Correct :)

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