TBS vs. PBS fluorescence

[Guillermo Palchik]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I have been trying to locate a paper I had read a few years ago  
regarding the use of TBS rather than PBS for IHC, since it reduces the  
level of background fluorescence associated with the PBS. Does anybody  
know what paper I am referring to?
In addition, does anybody have any insight into whether this is  
actually true? I mean, if I do my IHC with PBS and then wash it off  
with dH20, isn't this getting rid of most of the PBS background  
fluorescence?

Thanks,

Gil Palchik

[hidden email]

[Kurt Thorn]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I can't see how PBS can contribute any fluorescence at all.  Neither
phosphate nor NaCl should be fluorescent at all, and I've routinely used
phosphate buffers for fluorimetry measurements.

Kurt

Guillermo Palchik wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Confocalists,
>
> I have been trying to locate a paper I had read a few years ago
> regarding the use of TBS rather than PBS for IHC, since it reduces the
> level of background fluorescence associated with the PBS. Does anybody
> know what paper I am referring to?
> In addition, does anybody have any insight into whether this is
> actually true? I mean, if I do my IHC with PBS and then wash it off
> with dH20, isn't this getting rid of most of the PBS background
> fluorescence?
>
> Thanks,
>
> Gil Palchik
>
> [hidden email]
>

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone [phone]
fax   [phone]

[anh2006]

I don't know about autofluorescence but I do understand that antibodies, especially mAbs, may bind to antigen better in TBS. I believe I read this in a DAKO staining manual many years back.


------Original Message------
From: Guillermo Palchik
Sender: Confocal Microscopy List
To: [hidden email]
ReplyTo: Confocal Microscopy List
Sent: Aug 4, 2008 2:34 PM
Subject: TBS vs. PBS fluorescence

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I have been trying to locate a paper I had read a few years ago  
regarding the use of TBS rather than PBS for IHC, since it reduces the  
level of background fluorescence associated with the PBS. Does anybody  
know what paper I am referring to?
In addition, does anybody have any insight into whether this is  
actually true? I mean, if I do my IHC with PBS and then wash it off  
with dH20, isn't this getting rid of most of the PBS background  
fluorescence?

Thanks,

Gil Palchik

[hidden email]

[Beat Ludin]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Gil

PBS is not significantly fluorescent AFAIK. If I remember correctly
the amine group of the Tris saturates (fixation-induced) reactive
groups on the sample which would have a tendency to form fluorescent
moieties otherwise. So PBS doesn't induce background fluorescence,
but TBS reduces it.
Also, Tris should be used when when alkaline phosphatase-conjugated
antibodies are used for stainings.

Beat

At 20:34 04-08-2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Confocalists,
>
>I have been trying to locate a paper I had read a few years ago
>regarding the use of TBS rather than PBS for IHC, since it reduces the
>level of background fluorescence associated with the PBS. Does anybody
>know what paper I am referring to?
>In addition, does anybody have any insight into whether this is
>actually true? I mean, if I do my IHC with PBS and then wash it off
>with dH20, isn't this getting rid of most of the PBS background
>fluorescence?
>
>Thanks,
>
>Gil Palchik
>
>[hidden email]

[Guillermo Palchik]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you all so much for all your help!
Gil


On Aug 4, 2008, at 3:03 PM, Beat Ludin wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Gil
>
> PBS is not significantly fluorescent AFAIK. If I remember correctly  
> the amine group of the Tris saturates (fixation-induced) reactive  
> groups on the sample which would have a tendency to form fluorescent  
> moieties otherwise. So PBS doesn't induce background fluorescence,  
> but TBS reduces it.
> Also, Tris should be used when when alkaline phosphatase-conjugated  
> antibodies are used for stainings.
>
> Beat
>
> At 20:34 04-08-2008, you wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear Confocalists,
>>
>> I have been trying to locate a paper I had read a few years ago
>> regarding the use of TBS rather than PBS for IHC, since it reduces  
>> the
>> level of background fluorescence associated with the PBS. Does  
>> anybody
>> know what paper I am referring to?
>> In addition, does anybody have any insight into whether this is
>> actually true? I mean, if I do my IHC with PBS and then wash it off
>> with dH20, isn't this getting rid of most of the PBS background
>> fluorescence?
>>
>> Thanks,
>>
>> Gil Palchik
>>
>> [hidden email]

Guillermo Palchik
[hidden email]