dI loading of brain slices

[Kristen O'Connell]

This isn't strictly a confocal question, but I'm hoping someone here can
help or point me in the right direction...

A colleague wants to label olfactory bulb slices with dI and is having
difficulty getting the label to incorporate.  She is using the dI paste and
has already tried diluting it in DMSO and loading using a needle with no
success -- the dI does not incorporate into the membrane.

Has anyone here done this?  Any tips on how to make it work?

Kristen

[xavier Sanjuan]

Hi Kristen,

I have users which have successfully loaded DiI (I guess you refer to this
when you write dI) into 100 µm thick brain slices using a gene gun. The
tricky part is to get the optimal distance to shoot and the pore diameter of
the filter you put in front of the slices to avoid overloading, but they
needed only few attempts to get the correct protocol.

Hope this helps,

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
08003 Barcelona - Spain

Nou telèfon:  + 34 93 316 02 06

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


-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de Kristen O'Connell
Enviado el: sábado, 12 de junio de 2010 18:36
Para: [hidden email]
Asunto: dI loading of brain slices

This isn't strictly a confocal question, but I'm hoping someone here can
help or point me in the right direction...

A colleague wants to label olfactory bulb slices with dI and is having
difficulty getting the label to incorporate.  She is using the dI paste and
has already tried diluting it in DMSO and loading using a needle with no
success -- the dI does not incorporate into the membrane.

Has anyone here done this?  Any tips on how to make it work?

Kristen

[Bjorn Tyrberg]

A colleague with lot of DiI experience says:
You should use crystals. DMSO solution is mostly used for live labeling through
injections. Place a tiny crystal in the slice where the neurons of interest are
situated. DiI will incorporate into the membranes and transport along axons.
Use fine forceps or syringes to make incision and place crystal. Note that DiI
is sticky and a lot of care has to be taken to be precise. A good scope is a
must.
 
Bjorn

On Sat, 12 Jun 2010 11:36:08 -0500, Kristen O'Connell
<[hidden email]> wrote:

>This isn't strictly a confocal question, but I'm hoping someone here can
>help or point me in the right direction...
>
>A colleague wants to label olfactory bulb slices with dI and is having
>difficulty getting the label to incorporate.  She is using the dI paste and
>has already tried diluting it in DMSO and loading using a needle with no
>success -- the dI does not incorporate into the membrane.
>
>Has anyone here done this?  Any tips on how to make it work?
>
>Kristen

[Kristen O'Connell]

Thanks.  I'll pass this along.

I did mean DiI, not dI, obviously still hadn't had enough coffee when  
I wrote that!


On Jun 15, 2010, at 7:54 AM, Bjorn Tyrberg wrote:

> A colleague with lot of DiI experience says:
> You should use crystals. DMSO solution is mostly used for live  
> labeling through
> injections. Place a tiny crystal in the slice where the neurons of  
> interest are
> situated. DiI will incorporate into the membranes and transport  
> along axons.
> Use fine forceps or syringes to make incision and place crystal.  
> Note that DiI
> is sticky and a lot of care has to be taken to be precise. A good  
> scope is a
> must.
>
> Bjorn
>
> On Sat, 12 Jun 2010 11:36:08 -0500, Kristen O'Connell
> <[hidden email]> wrote:
>
>> This isn't strictly a confocal question, but I'm hoping someone  
>> here can
>> help or point me in the right direction...
>>
>> A colleague wants to label olfactory bulb slices with dI and is  
>> having
>> difficulty getting the label to incorporate.  She is using the dI  
>> paste and
>> has already tried diluting it in DMSO and loading using a needle  
>> with no
>> success -- the dI does not incorporate into the membrane.
>>
>> Has anyone here done this?  Any tips on how to make it work?
>>
>> Kristen

------------------------------------------------
Kristen O'Connell, Ph.D.
Assistant Professor
Department of Physiology
University of Tennessee Health Science Center
Memphis TN 38163
[phone] (voice)
[phone] (fax)
[hidden email]

[Jerry (Gerald) Sedgewick]

Here's why the 1st Annual Imaging in Research Course is being given in August, 2010:

* Visual data in the form of images is often 50 percent of a manuscript's content.  It's content is a reflection of the lab, and so the quality and accuracy of images is as important as the written content. 

* 44 percent of cases investigated by the Office of Research Integrity in 2005-6 involved accusations of image fraud, compared with about 6 percent a decade before that (1). These cases are rising, and most involve graduate and post-doctoral students. 

* Students and staff who self-report familiarity with imaging programs like Photoshop may be using it improperly.

In response to this, the "1st Annual Imaging in Research Course: Ethics, Acquisition, Post-Processing, Output and Segmenting" is being held at the University of Minnesota Continuing Education Center in Minneapolis/St. Paul, Minnesota, August 16 - 19, 2010, sponsored by the Histochemical Society and the Adobe Corporation.  Attendees can choose to attend the course for 1- to 4-days.  This workshop will educate those in science, medicine and engineering about correct techniques when acquiring, post-processing, and adjusting images for outputs; along with techniques that work for segmenting complex, biological images (for subsequent image analysis).

Other benefits of taking the course will likely result in:

Faster acceptance of submitted manuscripts
Authors better able to demonstrate outcomes to their target audience
Faster results from quantitation, with improved ability to segment desired features
Better documentation of imaging procedures
Standardization of post-processing
Learning to adjust and modify images minimally and through the objective use of numbers.

Jerry Sedgewick will present, along with invited speakers.  Jerry directed a core light microscopy and imaging facility for 15 years at the University of Minnesota, published 2 books on Photoshop and digital imaging, and his quantitative work has led to FDA approval for start up companies.

Please go to http://www.imagingandanalysis.com/seminars.html for more information.  There is a limit of 30 seats.

The cost ranges from $195 for 1-day to $840 for 4-days. It includes lunch, beverages and snacks.  Registrations for those who received information about this course via their core facility will receive discounts. Here's the summary for the days:

Day1: Ethics of digital imaging, sample preparation, calibration, best acquisition practices on light microscopes.
Day2: Post Processing I: setting up Photoshop, opening image stacks/12 bit images, rotate/crop, uneven illumination correction, color correction, histogram (tone) matching, gamma corrections, correcting noise, scale bars, extended focus, extended dynamic range, pseudocolor, tonal adjustment.  These functions also covered for the free programs GIMP and Image J (when applicable).
Day 3: Post Processing II: De-colorizing/colorizing fluorescent samples, merging images, colocalization, adjusting tones for 3D reconstructions, saving images, resetting pixel resolution (resampling), creating automated steps (macros), image stitching, making figures, better methods for sharpening, digital video, images to various outputs. These functions also covered for the free programs GIMP and Image J (when applicable).
Day 4: Segmenting in Photoshop for image analysis (quantitation): optical density and intensity measurements, creating binary files through 3 methods, setting threshold at consistent value, unbiased sampling (stereology), automating steps, measurement in Image J and Excel.

All the best,

Jerry Sedgewick

1. "Journals Find Many Images in Research Are Faked,"
Jeffrey R. Young, The Chronicle of Higher Education, September 9, 2009)