CYTO2011 Pre-congress courses announcement - Fundamentals of Image Cytometry before ISAC 2011 in Baltimore

[George McNamara]

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Dear Confocal listservites,

You may find of interest the Fundamentals of Image Cytometry 1.5 day
course before ISAC 2011 in Baltimore.

Hope to see you at the course and/or the meeting,
Sincerely,
George


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*CYTO2011 Pre-congress Courses, May 19^th -20^th , 2011*

*http://tinyurl.com/cyto2011courses*


*Fundamentals of Image Cytometry* <http://tinyurl.com/cyto2011courses>
Modern optical microscopy provides powerful tools for studying
biological samples varying from sub‐cellular structures through cells to
tissues. Effective use of these techniques, especially in the context of
image cytometry in which the quantitative aspect is of highest
importance, requires understanding not only optics, cell biology, and
biochemistry, but also image processing, pattern recognition, and
analysis. The goal of this short course is to provide that necessary
background and introduce the terminology, concepts, and methodological
approaches employed in modern image cytometry and high‐content
screening. The course is intended for researchers and students who have
previous experience with other optical methods such as flow cytometry or
basic optical microscopy. It will provide participants with the required
tools for quantitative interpretation of image‐based experiments. The
course will expand and strengthen the participants’ knowledge and
understanding of modern digital high‐content microscopy techniques. It
will stress the quantitative nature of imaging and draw parallels
between image pattern‐analysis techniques and flow cytometry data analysis.
The course does not require a thorough background in mathematics,
physics, or statistics. However; a very basic understanding of
fluorescence‐based techniques and cell biology will be assumed.  A full
course description and schedule can be found here
<http://www.cytoconference.org/pages/pre-congress-course.aspx>.

*Fundamentals of Flow Cytometry* <http://tinyurl.com/cyto2011courses>
This course will focus on the fundamentals of flow cytometry theory,
hardware and applications, and will cover basic topics such as light
scatter and fluorescence to advanced topics including cell sorting,
compensation, data analysis and biosafety. This course offers the
opportunity to interact with our international faculty and illustrious
mentors, along with many vendor demonstrations of the latest cytometers
and software. A full course description and schedule can be found here
<http://www.cytoconference.org/pages/pre-congress-course.aspx>.

*Building Rainbows**: **Fundamentals of PolychromaticFlow Cytometry*
<http://tinyurl.com/cyto2011courses>
Polychromatic flow cytometry has matured tremendously in recent years,
providing a means to study single cells in unprecedented detail.  The
building blocks for this technology (state-of-the-art flow cytometers,
reagents, and data analysis software) have become increasingly
accessible; however, effective use of these tools requires careful
experimental design and execution. This course, taught by experts in the
field, describes the fundamentals of polychromatic flow cytometry, and
provides strategies to synthesize this information into successful
polychromatic experiments.  It is recommended for intermediate-level
users. A full schedule can be found here
<http://www.cytoconference.org/pages/pre-congress-course.aspx>.

Register today (with or without full CYTO2011 participation) at
http://www.cytoconference.org/pages/registration.aspx

Thank you and we look forward to seeing you in Baltimore.

/Awtar Krishan, Zosia Maciorowski, Co-Chairs, Education Committee / /
Pratip K. Chattopadhyay, Coordinator Polychromatic Flow Cytometry Course / /
Rachel Walker, Coordinator Flow Course //
//Bartek Rajwa, Coordinator Image Course/

[DamirSudar]

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Dear all,

I would really appreciate some good suggestions how to keep
immunofluorescently labeled slides of mammalian cells good for long
periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
freezing at -20 or -80 a good idea? Any preparation tricks that keep the
fluorescence and the localization intact?

Thanks,
- Damir

--
Damir Sudar - Staff Scientist and Deputy for Technology
Lawrence Berkeley Laboratory / Life Sciences Division
One Cyclotron Road, MS 977R225A, Berkeley, CA 94720, USA
T: 510/486-5346 - F: 510/486-5586 - E: [hidden email]
WWW: http://www.lbl.gov/lifesciences/labs/sudar_lab.html

[Greg Martin-8]

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Hey Damir et.al. --

    Fix your preps briefly with PFA after the final post-secondary rinse.  Loss of signal in storage is usually due to the antibodies coming off.  A quick fix will help to "lock" the antibodies in place.

Be peace!  Greg.

Greg Martin

Keck Microscopy Facility
University of Washington School of Medicine
www.depts.washington.edu/keck

206-685-8784 (office)
425-344-2632 (cell)
----- Original Message -----
From: "Damir Sudar" <[hidden email]>
To: <[hidden email]>
Sent: Tuesday, May 03, 2011 10:58 AM
Subject: Keeping IMF slides good for long period?

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I would really appreciate some good suggestions how to keep
> immunofluorescently labeled slides of mammalian cells good for long
> periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
> freezing at -20 or -80 a good idea? Any preparation tricks that keep the
> fluorescence and the localization intact?
>
> Thanks,
> - Damir
>
> --
> Damir Sudar - Staff Scientist and Deputy for Technology
> Lawrence Berkeley Laboratory / Life Sciences Division
> One Cyclotron Road, MS 977R225A, Berkeley, CA 94720, USA
> T: 510/486-5346 - F: 510/486-5586 - E: [hidden email]
> WWW: http://www.lbl.gov/lifesciences/labs/sudar_lab.html
>

[Michael Schell]

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This is what works best for us:

1.  Mount in Prolong antifade with no air bubbles between the slide and coverslip.  Dry about 24 hr at RT.
2.  Seal edges with ample amounts of nail polish and allow to dry.
3.  Clean slides with a few drops of water to clean off salt, grime, and excess Prolong before storing flat, dark at 4 degrees.
4.  We find that having immersion oil on the slide for prolonged periods sometimes promotes the degradation of the fluorphores.  Rather than storing the slides with the oil on them, we blot off the excess with a Kimwipe, then do a quick clean with 70% ethanol, followed by a wipe with distilled water before returning to the flat storage at 4 degrees.  If some of the nail polish flakes off, we re-seal at this point.

Using this routine, we've had slides last as long as 5 years.  Not all will last this long, and the variable is probably the amount of oxygen that get into the sealed slide (or was there to begin with, owing to trapped air bubbles).

Mike

Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> Damir Sudar <[hidden email]> 05/03/11 1:58 PM >>>
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Dear all,

I would really appreciate some good suggestions how to keep
immunofluorescently labeled slides of mammalian cells good for long
periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
freezing at -20 or -80 a good idea? Any preparation tricks that keep the
fluorescence and the localization intact?

Thanks,
- Damir

--
Damir Sudar - Staff Scientist and Deputy for Technology
Lawrence Berkeley Laboratory / Life Sciences Division
One Cyclotron Road, MS 977R225A, Berkeley, CA 94720, USA
T: 510/486-5346 - F: 510/486-5586 - E: [hidden email]
WWW: http://www.lbl.gov/lifesciences/labs/sudar_lab.html

Classification:  UNCLASSIFIED
Caveats: None


Classification:  UNCLASSIFIED
Caveats: None

[Martin Wessendorf-2]

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Dear Damir--

On 5/3/2011 12:58 PM, Damir Sudar wrote:

> I would really appreciate some good suggestions how to keep
> immunofluorescently labeled slides of mammalian cells good for long
> periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
> freezing at -20 or -80 a good idea? Any preparation tricks that keep the
> fluorescence and the localization intact?

We stain tissue sections but I think our method should work for cells as
well.  We stain with Cy2, Cy3, rhodamine and Cy5-labeled secondaries
(and in the old days, Bodipy) and/or DNA dyes--but NOT fluorescein.
After staining, we dehydrate the tissue in graded alcohols, clear in
xylene and mount with DPX.  The DNA dyes tend to photobleach but
photobleaching of the secondaries is acceptably low for our purposes.

I have fluorescently stained sections that I processed that way almost
20 years ago and have kept on my desk top in the intervening time, that
I still use for teaching purposes.  I like having a solid mounting
medium that doesn't require cold-storage.

Good luck!

Martin Wessendorf


--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Boswell, Carl A - (cboswell)]

Freezing (-20C) seems to work fine with all the preps I've seen in which this has been done.  It probably helps that the antifade mounting medium has glycerol in it.  Sealing the coverslip with nail polish or other hardening medium helps to limit dehydration, as well as secure it when cleaning or using under oil.  I've seen very nice neuromuscular junction staining (standard immunostaining protocols) that were several months old.
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, May 03, 2011 12:07 PM
To: [hidden email]
Subject: Re: Keeping IMF slides good for long period?

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Dear Damir--

On 5/3/2011 12:58 PM, Damir Sudar wrote:

> I would really appreciate some good suggestions how to keep
> immunofluorescently labeled slides of mammalian cells good for long
> periods (3-5 months). The fluors are typically Alexa dyes or similar.
> Is freezing at -20 or -80 a good idea? Any preparation tricks that
> keep the fluorescence and the localization intact?

We stain tissue sections but I think our method should work for cells as well.  We stain with Cy2, Cy3, rhodamine and Cy5-labeled secondaries (and in the old days, Bodipy) and/or DNA dyes--but NOT fluorescein.
After staining, we dehydrate the tissue in graded alcohols, clear in xylene and mount with DPX.  The DNA dyes tend to photobleach but photobleaching of the secondaries is acceptably low for our purposes.

I have fluorescently stained sections that I processed that way almost
20 years ago and have kept on my desk top in the intervening time, that I still use for teaching purposes.  I like having a solid mounting medium that doesn't require cold-storage.

Good luck!

Martin Wessendorf


--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Stephen Cody-2]

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Dear Damir,

I can second Martin's suggestion. I've had Alexa488, Alexa546 and Cy3 (not
on the same slide) antibody stained cultured cells mounted in DPX for over
20 years. I just keep them in a box so the sun doesn't shine on them. You
may need to dehydrate gently to prevent cells flattening out, and infiltrate
with xylene prior to mounting. The cells were 4% PFA fixed.

If you need a reference (there may be others):
Cody, S.H., Williams, D.A. Optimizing confocal microscopy for thick
biological specimens. *In: Fluorescent and luminescent probes for biological
activity - A practical guide to technology for quantitative real-time
analysis* (ed W.T. Mason) Academic Press Limited, U.K. 2nd edition. Chapter
27 pp 350- 360. (1999).

Cheers
Steve


On 4 May 2011 05:07, Martin Wessendorf <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Damir--
>
>
> On 5/3/2011 12:58 PM, Damir Sudar wrote:
>
> I would really appreciate some good suggestions how to keep
>> immunofluorescently labeled slides of mammalian cells good for long
>> periods (3-5 months). The fluors are typically Alexa dyes or similar. Is
>> freezing at -20 or -80 a good idea? Any preparation tricks that keep the
>> fluorescence and the localization intact?
>>
>
> We stain tissue sections but I think our method should work for cells as
> well.  We stain with Cy2, Cy3, rhodamine and Cy5-labeled secondaries (and in
> the old days, Bodipy) and/or DNA dyes--but NOT fluorescein. After staining,
> we dehydrate the tissue in graded alcohols, clear in xylene and mount with
> DPX.  The DNA dyes tend to photobleach but photobleaching of the secondaries
> is acceptably low for our purposes.
>
> I have fluorescently stained sections that I processed that way almost 20
> years ago and have kept on my desk top in the intervening time, that I still
> use for teaching purposes.  I like having a solid mounting medium that
> doesn't require cold-storage.
>
> Good luck!
>
> Martin Wessendorf
>
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>

--
Stephen H. Cody