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Carl has shared some useful information in previous posts about where to get started with cryosectioning, and important points to consider. I have also found the following two websites helpful regarding cryosectioning:
1) http://neuromuscular.wustl.edu/lab/freeze.htm This website gives useful information on how to freeze the muscle after harvest. I have tried several different methods, including the ones that Carl described, so you will surely have to figure out which one will work best for you.
I'm sure you know, and I have found out that it takes a long time to optimize someone else's protocol to your own experimental conditions. There are so many different factors and variables to consider that you probably will need to piece together different protocols to get results that you are satisfied with.
Mon Jan 11, 2010 9:37 pm
JonA
Rank: Member
Joined: Dec 09, 2009
Posts: 8
Post subject:
I have been continuing with trying to optimize conditions for the muscle and antibodies I am working with. I uploaded two pictures of the same section, but focused on different planes. I seem to be getting these cloudy or foggy areas in the sections. Does anyone know how to prevent these from occuring? Is it still a fixation problem? They seem to occur regardless of air dry or no air dry. I am cutting at 8 micron thickness, would a thinner or thicker section help? Thanks again for your help.
Many thanks for your informative contributions...sincerely!
Sorry that you are STILL frustrated.
Seems to me that the muscle sections are lifting/contracting.
It is also frustrating to us as we can't be there to try to sort out the problem ( so many problems absolutely require on-site help/discussion that I am often embarrassed by my inability to help).
Anyway, less of the self-flagellation
Please, can you stain one section with H&E and post?
It may help.
For every Immunostain, I will include a H&E because it can give so much more information.
I note that Histonet has a request for a way to get flatter sections for LSM....well, for LSM, they don't need to be flatter but...is the person asking how to get rid of wrinkles in the section??
Sorry, JonA...you have been great re images.
Have you stained your sections for MHC/Desmin and stained the nuclei with DAPI/Hoechst?
If I see a Hoechst image I can tell if it is out of focus: the images you present are out of focus, which only indicates, to me, that your image capture system is not optimised.
Sorry to not have a Magic wand.
Car
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