DAPI bleaching issue

[xavier Sanjuan]

Hi list,

 

One of my users has very quick DAPI bleaching when observing her samples, wich sometimes make even difficult to get images. Most of my users use routinely DAPI in their samples and never had such a problem.

 

She is performing a immunofluorescence with the tyramide kit from Perin Elmer (peroxidase blocking step, blocking with TNB, whashing steps with TNT) on muscle mouse sections (10 µm thick approx). She is using Acetone as a fixative (10 min), and is mounting with Vectashield after DAPI staining (10 min).

 

DAPI observation and imaging is performed on a Leica DM6000B with a standard blue fluorescence filter and the Hg lamp set at minimum power (10%). As I said, over the years tens of users have imaged DAPI staining on a lot of different cells/tissues using the same setup and never observed such a quick bleaching.

 

Her protocol seems quite standard for me, but I have little experience both with acetone as a fixative and with tyramide amplification, so perhaps I am missing something. Also I have read in the list some issues with DAPI with Vectashield.

 

Any advice/tip will be welcome. Feel free to contact me off list if you need the full protocol to make a better diagose.

 

Thank you very much in advance!

 

Best,

 

Xavi.

 

___________________________________

Xavier Sanjuan
Servei de Microscòpia Confocal
Departament de Ciències Experimentals i de la Salut
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 - Sala 309
08003 Barcelona - Spain

Tel.:  + 34 93 316 08 64
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web: http://www.upf.edu/sct

 

[Craig Brideau]

How's her dye concentration compare to other users?  Is there more oxygen around in the sample somehow?  Is something wrong with the Vectashield prep?

Just some ideas we came up with 'around the water cooler'...

Craig

On Tue, Jan 20, 2009 at 4:50 AM, Xavier Sanjuan <[hidden email]> wrote:

Hi list,

 

One of my users has very quick DAPI bleaching when observing her samples, wich sometimes make even difficult to get images. Most of my users use routinely DAPI in their samples and never had such a problem.

 

She is performing a immunofluorescence with the tyramide kit from Perin Elmer (peroxidase blocking step, blocking with TNB, whashing steps with TNT) on muscle mouse sections (10 µm thick approx). She is using Acetone as a fixative (10 min), and is mounting with Vectashield after DAPI staining (10 min).

 

DAPI observation and imaging is performed on a Leica DM6000B with a standard blue fluorescence filter and the Hg lamp set at minimum power (10%). As I said, over the years tens of users have imaged DAPI staining on a lot of different cells/tissues using the same setup and never observed such a quick bleaching.

 

Her protocol seems quite standard for me, but I have little experience both with acetone as a fixative and with tyramide amplification, so perhaps I am missing something. Also I have read in the list some issues with DAPI with Vectashield.

 

Any advice/tip will be welcome. Feel free to contact me off list if you need the full protocol to make a better diagose.

 

Thank you very much in advance!

 

Best,

 

Xavi.

 

___________________________________

Xavier Sanjuan
Servei de Microscòpia Confocal
Departament de Ciències Experimentals i de la Salut
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 - Sala 309
08003 Barcelona - Spain

Tel.:  + 34 93 316 08 64
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web: http://www.upf.edu/sct

 

[Lingle, Wilma L., Ph.D.]

We have observed that after acetone or methanol fixation, nuclei lose integrity over a relatively short period of time.  Perhaps she is observing that rather than photobleaching.  We solved the problem by doing a very short (para)formaldehyde fixation of the tissue on the slide after immunolabeling and just prior to DAPI incubation.

 

Wilma Lingle

Wilma L. Lingle, Ph.D.
Director, TACMA Shared Resource
Mayo Clinic Cancer Center
[phone]
[hidden email]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, January 20, 2009 2:58 PM
To: [hidden email]
Subject: Re: DAPI bleaching issue

How's her dye concentration compare to other users?  Is there more oxygen around in the sample somehow?  Is something wrong with the Vectashield prep?

Just some ideas we came up with 'around the water cooler'...

Craig

On Tue, Jan 20, 2009 at 4:50 AM, Xavier Sanjuan <[hidden email]> wrote:

Hi list,

 

One of my users has very quick DAPI bleaching when observing her samples, wich sometimes make even difficult to get images. Most of my users use routinely DAPI in their samples and never had such a problem.

 

She is performing a immunofluorescence with the tyramide kit from Perin Elmer (peroxidase blocking step, blocking with TNB, whashing steps with TNT) on muscle mouse sections (10 µm thick approx). She is using Acetone as a fixative (10 min), and is mounting with Vectashield after DAPI staining (10 min).

 

DAPI observation and imaging is performed on a Leica DM6000B with a standard blue fluorescence filter and the Hg lamp set at minimum power (10%). As I said, over the years tens of users have imaged DAPI staining on a lot of different cells/tissues using the same setup and never observed such a quick bleaching.

 

Her protocol seems quite standard for me, but I have little experience both with acetone as a fixative and with tyramide amplification, so perhaps I am missing something. Also I have read in the list some issues with DAPI with Vectashield.

 

Any advice/tip will be welcome. Feel free to contact me off list if you need the full protocol to make a better diagose.

 

Thank you very much in advance!

 

Best,

 

Xavi.

 

___________________________________

Xavier Sanjuan
Servei de Microscòpia Confocal
Departament de Ciències Experimentals i de la Salut
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 - Sala 309
08003 Barcelona - Spain

Tel.:  + 34 93 316 08 64
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web: http://www.upf.edu/sct

 

[Elizabeth Nickless]

Are you dual staining at all?  I have had problems with photobleaching of some probes when trying variable concentrations of  probe mixes. And have found that  increasing concentrations of one probe over another can cause a result of photobleaching of other probes in the mix.

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lingle, Wilma L., Ph.D.
Sent: Wednesday, 21 January 2009 11:14 a.m.
To: [hidden email]
Subject: Re: DAPI bleaching issue

We have observed that after acetone or methanol fixation, nuclei lose integrity over a relatively short period of time.  Perhaps she is observing that rather than photobleaching.  We solved the problem by doing a very short (para)formaldehyde fixation of the tissue on the slide after immunolabeling and just prior to DAPI incubation.

 

Wilma Lingle

Wilma L. Lingle, Ph.D.
Director, TACMA Shared Resource
Mayo Clinic Cancer Center
[phone]
[hidden email]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, January 20, 2009 2:58 PM
To: [hidden email]
Subject: Re: DAPI bleaching issue

How's her dye concentration compare to other users?  Is there more oxygen around in the sample somehow?  Is something wrong with the Vectashield prep?

Just some ideas we came up with 'around the water cooler'...

Craig

On Tue, Jan 20, 2009 at 4:50 AM, Xavier Sanjuan <[hidden email]> wrote:

Hi list,

 

One of my users has very quick DAPI bleaching when observing her samples, wich sometimes make even difficult to get images. Most of my users use routinely DAPI in their samples and never had such a problem.

 

She is performing a immunofluorescence with the tyramide kit from Perin Elmer (peroxidase blocking step, blocking with TNB, whashing steps with TNT) on muscle mouse sections (10 µm thick approx). She is using Acetone as a fixative (10 min), and is mounting with Vectashield after DAPI staining (10 min).

 

DAPI observation and imaging is performed on a Leica DM6000B with a standard blue fluorescence filter and the Hg lamp set at minimum power (10%). As I said, over the years tens of users have imaged DAPI staining on a lot of different cells/tissues using the same setup and never observed such a quick bleaching.

 

Her protocol seems quite standard for me, but I have little experience both with acetone as a fixative and with tyramide amplification, so perhaps I am missing something. Also I have read in the list some issues with DAPI with Vectashield.

 

Any advice/tip will be welcome. Feel free to contact me off list if you need the full protocol to make a better diagose.

 

Thank you very much in advance!

 

Best,

 

Xavi.

 

___________________________________

Xavier Sanjuan
Servei de Microscòpia Confocal
Departament de Ciències Experimentals i de la Salut
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 - Sala 309
08003 Barcelona - Spain

Tel.:  + 34 93 316 08 64
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web: http://www.upf.edu/sct

 

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