Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Cy7-TSA-dye-tp7587083p7587090.html
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Hi again Sripad,
a do-it-yourself conjugation method of NHS-fluorophore and tyramine is in:
Each GPI-anchored protein species forms a specific lipid raft depending
on its GPI attachment signal.
Miyagawa-Yamaguchi A,*Kotani N*,*Honke*K.
Glycoconj J. 2015 Oct;32(7):531-40. doi: 10.1007/s10719-015-9595-5. Epub
2015 May 7.
PMID:
25948169
We previously reported a method, termed enzyme-mediated activation of
radical sources (EMARS) for analysis of co-clustered molecules with
horseradish peroxidase (HRP) fusion proteins expressed in living cells.
This method is featured by radical formation of*labeling*reagents by
HRP. In the current study, we have employed another*labeling*reagent,
fluorescein-conjugated*tyramide*(FT) instead of the original arylazide
compounds. Although hydrogen peroxide is required for the activation of
FT, the*labeling*efficiency by HRP and the nonspecific reactions by
endogenous enzyme(s) have been dramatically improved compared with the
original fluorescein arylazide. This revised EMARS method has enabled
visualization of co-clustered molecules in the endoplasmic reticulum and
Golgi membranes with confocal microscopy. By using this method, we have
found that GPI-anchored proteins, decay accelerating factor (DAF) and
Thy-1 are exclusively co-clustered with HRP-DAFGPI and HRP-Thy1GPI, in
which GPI attachment signals of DAF and Thy-1 have been connected to
HRP, respectively. Furthermore, the N-glycosylation types of DAF and
Thy-1 have been found to correspond to those of HRP-DAFGPI and
HRP-Thy1GPI, respectively. These results indicate that each GPI-anchored
protein species forms a specific lipid raft depending on its GPI
attachment signal, and that the EMARS method can segregate individual
lipid rafts.
KEYWORDS:
EMARS; Fluorescein*tyramide*; HRP; N-glycosylation; Visualization of
co-clustering molecules
PMID:
25948169 DOI: 10.1007/s10719-015-9595-5
Synthesis of fluorescein-conjugated tyramide
100 mg of tyramine (Sigma) dissolved in 5 mL N,Ndimethylacetamide (DMAA)
was added to 100 mg NHS fluorescein (Thermo) in 5 mL DMAA. The mixture
was incubated at roomtemperature for 12 h in dark. An aliquot of the
mixture was applied to thin-layer chromatography
(chloroform:methanol:water = 65:25:4 vol/vol) to check whether
NHS-fluorescein was completely consumed. To remove excess tyramine, the
mixture was applied to 8 mL of cation-exchange resin, AG-50WX8 (Bio-Rad)
equilibrated with DMAA. An aliquot of the flow-through fraction was
applied to thin-layer chromatography again to check whether tyramine was
completely removed. The flow-through fraction containing
fluorescein-conjugated tyramine (FT) was stored at −20 °C.
//
Alexa Fluor 750 is the closest Alexa to Cy7 (see Semrock Searchlight or
Chroma Spectra Viewer for spectra). Many companies now offer NIR
fluorophores.
Alternatively, you could buy biotin-tyramide and AF750-streptavidin or
Cy7-streptavidin. Other indirect detection schemes exist, such as
digoxigenin-tyramide (find online or make your own) and
fluorophore-anti-DIG.
enjoy,
George
On 7/29/2017 10:05 AM, George McNamara wrote:
>
> Hi Sripad,
>
> Molecular Probes can conjugate whatever Alexa Fluor you want onto
> tyramide, and yes, there is an AF Cy7 equivalent.Tyramide is off
> patent, other 'chemistry' companies could conjugate their/your
> favorite fluorophores.
>
> //
>
> At higher plex, you are going to have spectral overlap: learn to love
> spectral unmixing. For example:
>
> PLoS One. 2016 Jul 8;11(7):e0158495. doi: 10.1371/journal.pone.0158495.
> eCollection 2016.
>
> Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.
>
> Valm AM(1)(2), Oldenbourg R(1)(2), Borisy GG(3).
>
> Author information:
> (1)Marine Biological Laboratory, Woods Hole, Massachusetts, United States of
> America.
> (2)Brown University, Providence, Rhode Island, United States of America.
> (3)Forsyth Institute, Cambridge, Massachusetts, United States of America.
>
> The number of fluorescent labels that can unambiguously be distinguished in a
> single image when acquired through band pass filters is severely limited by the
> spectral overlap of available fluorophores. The recent development of spectral
> microscopy and the application of linear unmixing algorithms to spectrally
> recorded image data have allowed simultaneous imaging of fluorophores with highly
> overlapping spectra. However, the number of distinguishable fluorophores is still
> limited by the unavoidable decrease in signal to noise ratio when fluorescence
> signals are fractionated over multiple wavelength bins. Here we present a
> spectral image analysis algorithm to greatly expand the number of distinguishable
> objects labeled with binary combinations of fluorophores. Our algorithm utilizes
> a priori knowledge about labeled specimens and imposes a binary label constraint
> on the unmixing solution. We have applied our labeling and analysis strategy to
> identify microbes labeled by fluorescence in situ hybridization and here
> demonstrate the ability to distinguish 120 differently labeled microbes in a
> single image.
>
> DOI: 10.1371/journal.pone.0158495
> PMCID: PMC4938436
> PMID: 27391327
>
>
> George
>
>
> On 7/28/2017 2:22 PM, S Ram wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>> Post images onhttp://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hello,
>> Just wondering if anyone has ever come across (or used) a Cy7 or similar
>> TSA dye probe? I checked a few of the vendors that I could think of
>> (Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had any
>> probes in that range.
>>
>> The goal is to do automated staining of FFPE tissues using Cy5 and another
>> NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
>> due to spectral overlap. Also, dyes in the visible region are no beuno due
>> to too much autofluorescence.
>>
>> Any help/suggestion is greatly appreciated.
>>
>> Thanks.
>>
>> Sripad
>
> --
>
>
> George McNamara, PhD
> Baltimore, MD 21231
>
[hidden email]
>
https://www.linkedin.com/in/georgemcnamara>
https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
>
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650>
http://confocal.jhu.edu (as of May 22, 2017)
>
--
George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamarahttps://works.bepress.com/gmcnamara/75/http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650