[Histonet] Re: muscle stain

[Andrea Marion]

Hi Saro and others,

Our protocol is attached. It is very simple.

The only trick is to remember that if you are using this for heart tissue,
each section will likely contain cardiomyocytes sectioned at various
angles. To accurately measure cell size/diameter, you will want to measure
only cells that are neatly cross-sectioned. If you measure cells that are
cross-sectioned at an angle, the measurements will be off. This is
discussed in the Dolber 1994 paper I mentioned. Essentially you want to
identify regions where the WGA staining is very crisp and thin, as this
will identify cells that are sectioned perpendicular to their long axis.
Cells that are cross-sectioned at some oblique angle will have a fuzzy
border of staining along one edge of the cell. This is sometimes hard to
identify (at least for me in mouse tissue), and should probably be
considered as a caveat to this technique. At the very least, I think it
needs to be considered and controlled for. If anyone has a better way of
identifying such cells, I'd be happy to hear it.

If you are using this technique on skeletal muscle (which I haven't
tried), I believe it would be much simpler as the myocytes are aligned
more regularly throughout the tissue, and it would be easy to orient the
tissue in the block in such a way that you get perfect cross-sectioned
myocytes.

Wheat Germ Agglutinin-FITC staining to measure myocyte size
1. Begin with 5-8 um FFPE sections on charged slides
2. Dewax slides
3. Rehydrate slides
4. Rinse in PBS 5 minutes x 3
5. Incubate 60 minutes with 100 ug/ml WGA-FITC in PBS + 1 mM CaCl2
6. Rinse in PBS 5 minutes x 3
7. Mount with Vectashield or Vectashield + DAPI
8. Image slides, collecting images of cross-sectioned myocytes
9. Measure area inside WGA-staining for perfectly cross-sectioned myocytes
using ImageJ

PBS: 8.5 mM Na2HPO4, 1.5 mM KH2PO4, 150 mM NaCl, pH 7.2
WGA-FITC: Vector Labs, Burlingame, CA  Product # FL-1021

Andrea Marion
Graduate Student
University of Illinois - Chicago
amario3 <at> uic <dot> edu


On Wed, August 18, 2010 8:47 am, Bascaramurty, Saro wrote:
> Hi Andrea,
>
> I wouldn't mind if you could email me your protocol.
>
> Thanks in advance.
>
> Saro Bascaramurty.
> IBD, NRC, Wpg. MB
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Andrea
> Marion
> Sent: August 18, 2010 8:19 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: muscle stain
>
> There is an immunofluorescence technique that uses wheat germ agglutinin
> (WGA) conjugated to a fluorophore (usually FITC). WGA binds only to the
> cell periphery, leaving you with beautiful traces of the outside of each
> cell. The cell area is then simple to analyze with ImageJ or any other
> software program. It's often used to measure cardiomyocyte size in
> hypertrophy studies. I can provide a protocol if requested.
>
> If you have access to this article, there is a detailed description of the
> technique and great images: Regional changes in myocyte structure in model
> of canine right atrial hypertrophy. Am J Physiol Heart Circ Physiol 267:
> H1279-H1287, 1994. P. C. Dolber, R. P. Bauman, J. C. Rembert and J. C.
> Greenfield Jr.
>
> If not, here is a freely accessible publication. Figure 9, panel B shows
> what the result looks like. Cardiomyocyte GATA4 functions as a
> stress-responsive regulator of angiogenesis in the murine heart. J Clin
> Invest. 2007; 117(11):3198.
> http://www.jci.org/articles/view/32573/figure/9
>
> Andrea Marion
> Graduate Student
> University of Illinois - Chicago
> amario3 <at> uic <dot> edu
>
>> To: histonet <@t> lists.utsouthwestern.edu
>> Subject: [Histonet] muscle stain
>>
>> I am posting this for a friend.  . They would like to stain muscle.
>> The eosin and hematoxylin stain gives us too much information since it
>> stains the nucleus separately from the cytoplasm and confuses the
>> image analysis software. What we need is the measurement of  the cell
>> diameter only, a simpler stain. Thanks for any info you can give me.
>> Deon Simon Any help you can give her on how to measure or a stain will
>> be very much appreciated.
>>
>> Margaret Perry HT(ASCP)
>> Dept of Veterinary and  Biomedical services Box 2175 South Dakota
>> State University Brookings SD 57007
>> 605-688-5638
>>
>
>
>
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>