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PerCP Cy5.5 for confocal

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anh2006

PerCP Cy5.5 for confocal

Dear All,

I need some advice in setting up for a very important experiment coming up. Has anyone used the fluorophore PerCP CY5.5 with success for confocal microscopy? Any experience with autofluorescence in this wavelength or whether this fluorophore is a severe bleacher etc.?

I want to do 5 or 6 color on a Zeiss LSM710 and am thinking about the following, I would love your feedback.

Alexa405
Alexa430
GFP
RFP
Alexa633
PerCP CY5.5

Thank you in advance,

Andrea

--------------------------------

Andrea T. Hooper, PhD

[hidden email]

G. Esteban Fernandez

Re: PerCP Cy5.5 for confocal

Alexa405 has been very dim and bleach-prone in my hands. I think if
you call Invitrogen they might tell you that "Marina blue" is a better
option, I remember Mike Ignatius posting something to that effect in
the past couple of years.

Good luck.

-Esteban

On Tue, Nov 3, 2009 at 4:16 PM, Andrea Hooper <[hidden email]> wrote:

> Dear All,
> I need some advice in setting up for a very important experiment coming up.
> Has anyone used the fluorophore PerCP CY5.5 with success for confocal
> microscopy? Any experience with autofluorescence in this wavelength or
> whether this fluorophore is a severe bleacher etc.?
> I want to do 5 or 6 color on a Zeiss LSM710 and am thinking about the
> following, I would love your feedback.
>
> Alexa405
> Alexa430
> GFP
> RFP
> Alexa633
> PerCP CY5.5
>
> Thank you in advance,
> Andrea
> --------------------------------
> Andrea T. Hooper, PhD
> [hidden email]

--
G. Esteban Fernandez, Ph.D.

Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO 65211

http://www.biotech.missouri.edu/mcc

(573)882-4895
(573)884-9676 fax

Junya HIROI

Re: PerCP Cy5.5 for confocal

In reply to this post by anh2006

Hi Andrea,

I actually did 5-color staining on a Zeiss LSM510, with
Alexa 405, Alexa 430, Alexa 488, Alexa 555 and Alexa 647.

http://jeb.biologists.org/cgi/content/full/211/16/2584
(Figs 6 and 8 are 5-color staining)

I used Alexa 405 and 430 in the above paper, but later I found that
Pacific Blue is better than Alexa 405, and Pacific Orange is better
than Alexa 430 (brighter and more stable, although not as stable as
other Alexa dyes, such as Alexa 488, 555 and 647). I did not check
Marina blue yet.

I did not compare Alexa 647 and Alexa 633, but I have heard that Alexa
647 is better than Alexa 633, even if with 633 nm laser.

Best wishes,

Junya

On 11/4/09, at 7:16 , Andrea Hooper wrote:

> Dear All,
>
> I need some advice in setting up for a very important experiment
> coming up. Has anyone used the fluorophore PerCP CY5.5 with success
> for confocal microscopy? Any experience with autofluorescence in
> this wavelength or whether this fluorophore is a severe bleacher etc.?
>
> I want to do 5 or 6 color on a Zeiss LSM710 and am thinking about
> the following, I would love your feedback.
> ? Alexa405
> ? Alexa430
> ? GFP
> ? RFP
> ? Alexa633
> ? PerCP CY5.5
> Thank you in advance,
> Andrea

___________________________________________
Junya HIROI
Department of Anatomy
St. Marianna University School of Medicine
2-16-1 Sugao, Miyamae-ku
Kawasaki 216-8511, JAPAN
phone: +81-44-977-8111 ext.3625
fax: +81-44-976-7083
e-mail: [hidden email]
___________________________________________

G. Esteban Fernandez

Re: PerCP Cy5.5 for confocal

It has been my experience that AlexaFluor647 is brighter than AF633
even with a 633 laser. The extinction coefficient of AF647 is more
than 2x that of AF633 so that compensates for the less-than-ideal
laser. In addition our standard "far red" emission filters better
match the emission spectrum of AF647 and I suspect the quantum yield
of AF647 is higher too, though the last time I checked the Invitrogen
website that specification was not given.

-Esteban

2009/11/4 Junya HIROI <[hidden email]>:

> Hi Andrea,
>
> I actually did 5-color staining on a Zeiss LSM510, with
> Alexa 405, Alexa 430, Alexa 488, Alexa 555 and Alexa 647.
>
> http://jeb.biologists.org/cgi/content/full/211/16/2584
> (Figs 6 and 8 are 5-color staining)
>
> I used Alexa 405 and 430 in the above paper, but later I found that Pacific
> Blue is better than Alexa 405, and Pacific Orange is better than Alexa 430
> (brighter and more stable, although not as stable as other Alexa dyes, such
> as Alexa 488, 555 and 647). I did not check Marina blue yet.
>
> I did not compare Alexa 647 and Alexa 633, but I have heard that Alexa 647
> is better than Alexa 633, even if with 633 nm laser.
>
> Best wishes,
>
> Junya
>
>
> On 11/4/09, at 7:16 , Andrea Hooper wrote:
>
>> Dear All,
>>
>> I need some advice in setting up for a very important experiment coming
>> up. Has anyone used the fluorophore PerCP CY5.5 with success for confocal
>> microscopy? Any experience with autofluorescence in this wavelength or
>> whether this fluorophore is a severe bleacher etc.?
>>
>> I want to do 5 or 6 color on a Zeiss LSM710 and am thinking about the
>> following, I would love your feedback.
>> ? Alexa405
>> ? Alexa430
>> ? GFP
>> ? RFP
>> ? Alexa633
>> ? PerCP CY5.5
>> Thank you in advance,
>> Andrea
>
> ___________________________________________
> Junya HIROI
> Department of Anatomy
> St. Marianna University School of Medicine
> 2-16-1 Sugao, Miyamae-ku
> Kawasaki 216-8511, JAPAN
> phone: +81-44-977-8111 ext.3625
> fax: +81-44-976-7083
> e-mail: [hidden email]
> ___________________________________________
>

Ignatius, Mike-2

Re: PerCP Cy5.5 for confocal

In reply to this post by G. Esteban Fernandez

Jason Kilgore, here at Probes, did a careful analysis a while back using Hg arc lamp illumination. We can't comment on what a 405 laser might do as we don't have one on our scopes.

For Brightness (Flow folks care about this the most)

Pacific BlueT > Marina Blue® > Alexa Fluor® 405.

But for photostability, which imagers care the most about:

w/out antifade

Marina Blue® > Alexa Fluor® 405 > Pacific BlueT

With our antifades, Prolong® Gold or Slowfade® gold

Marina Blue® >> Alexa Fluor® 405 > Pacific BlueT.

Thus Marina Blue® is preferred over the Alexa Fluor® 405 for imaging applications.

Mike Ignatius,

Molecular Probes/Invitrogen/lifetech

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez
Sent: Wednesday, November 04, 2009 6:07 AM
To: [hidden email]
Subject: Re: PerCP Cy5.5 for confocal

Alexa405 has been very dim and bleach-prone in my hands. I think if
you call Invitrogen they might tell you that "Marina blue" is a better
option, I remember Mike Ignatius posting something to that effect in
the past couple of years.

Good luck.

-Esteban

On Tue, Nov 3, 2009 at 4:16 PM, Andrea Hooper <[hidden email]> wrote: