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Hi Dale,
These are chemically different classes, so do not count on one method
working for all.
While not specifically mentioned by you, Alexa Fluor 647 is a Cy5
analog, should be destroyed by the Gerdes et al (GE multiOmyx) method, see
Highly multiplexed single-cell analysis of formalin-fixed,
paraffin-embedded cancer tissue.
Gerdes MJ, Sevinsky CJ, Sood A, Adak S, Bello MO, Bordwell A, Can A,
Corwin A, Dinn S, Filkins RJ, Hollman D, Kamath V, Kaanumalle S, Kenny
K, Larsen M, Lazare M, Li Q, Lowes C, McCulloch CC, McDonough E,
Montalto MC, Pang Z, Rittscher J, Santamaria-Pang A, Sarachan BD, Seel
ML, Seppo A, Shaikh K, Sui Y, Zhang J, Ginty F.
Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11982-7. doi:
10.1073/pnas.1300136110.
PMID: 23818604
and Gerdes patent.
May be simpler to just strip the antibodies off (be sure to wear PPE -
personal protective equipment -- while stripping antibodies).
See slides 11 and 12 of
https://digitalpathologyassociation.org/_data/files/2014_Pathology_Visions/PV14_Presentations/11_Day_2_Opening_Presentation_Levenson.pdf
for a first round 10plex. Also mentioned at
https://www.cacds.uh.edu/research/domain-applications/mapping-brain-tissue-alterations/
enjoy,
George
p.s. improving immunofluorescence on FFPE tissue sections - I encourage
checking out
Formaldehyde scavengers function as novel antigen retrieval agents.
Vollert CT, Moree WJ, Gregory S, Bark SJ, Eriksen JL.
Sci Rep. 2015 Nov 27;5:17322. doi: 10.1038/srep17322.
PMID: 26612041
and their corresponding patent (US 9506928,
http://www.freepatentsonline.com/9506928.html ), and to keep an eye on
the company, www.teomics.com, for product(s). I do not have a financial
connection to them (I met Craig V at JLabs@TMC events recently). They
do not present quantitation on how much these 'formaldehyde scavengers'
improve FFPE tissue sections antigen retrieval by -- my hope is 20x
improement for 80% of epitopes ... which I think would greatly improve
direct immunofluorescence, which in turn would enable multiplexing. Of
course I had high hopes for an A.R. method a decade ago (PubMed
20025485, 15637333) which did not catch on.
On 2/16/2017 10:12 AM, Dale Moulding wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
> does anyone know of a way to irreversibly destroy alexa fluor fluorescence so a sample can be stained with a second set of dyes?
> We do whole mount staining for 3 antigens with Alexa 488, 568 and 633. We would then like to re-image the same samples with cellmask and phalloidin.
> Is there any way to kill alexa fluor fluorescence without destroying membranes and phalloidin binding?
> Cheers
> Dale
--
George McNamara, PhD
Houston, TX 77054
[log in to unmask]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
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