Re: [Histonet] Re:Frozen Spleen w/bubbles

From:Maria Mejia

Hello, Jo Dee, I feel for you! I wish I could be there to see first hand 
this strange
(type) of bubbles on freshly cut cryo tissue sections.

 I'd like ask how the tissue (spleen) was initially frozen?

How does the frozen spleen surface look to you as you're cutting into it 
picking up any sections? Does the surface of the tissue look normal - by 
that I mean
does it look smooth?

You've probably have already surmised the reason for the tissue falling 
off - layer
/number of bubbles of air between the tissue and the glass surface of 
the slide.

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94103
Phone: (415)-345-2185

Jo Dee Fish wrote:

>> Dear Gayle,
> Thank you for your response, here are some answers to your questions. 
> In addition, the bubbles appear right after the section is picked up, 
> before any other process is performed, such as staining, IHC, 
> fixation, or coverslipping.  I am at a loss.  I don't see this problem 
> with any of my other tissues.
>> What species is the spleen from?
>> Mouse
>> How thick are the sections?
>> 10um
>> What temperature are you sectioning at?  i.e. knife temperature? 
>> Block/tissue temperature?
>> -20C
>> How to you pick up a FS onto the slide?  Slide to section so it comes 
>> onto glass surface, another method?
>> Glass to section.
>> What kind of blades are you using?
>> ThermoShandon MB35 Premier
>> Are you sections perfectly flat under antiroll device or by brush 
>> method, whichever you use?
>> Yes, nice and flat, I tried both methods on two different cryostats.
>> What kind of slides are you using?  Plus Charge?
>> Colorfrost Plus slides.
>> Before IHC do you air dry your sections at RT or overnight before 
>> fixation with what????
>> I'm not doing the IHC, the investigator is.  But anyway, I see the 
>> bubbles before anything is done to the slide, before fixation, 
>> drying, etc.
>> How are you fixing your spleens?
>> She has tried 3% paraformaldehyde, cold acetone, and zinc formalin.
>> Do you use a peroxidase blocker that has a high concentration of 
>> hydrogen peroxide?
>> I don't know what she is doing.  The sections came off of the slide 
>> regardless, whether or not IHC was performed.
> ********************************************************************** 
> **********
> Jo Dee Fish
> Research Technologist III
> Gladstone Institute of Cardiovascular Disease
> Telephone: (415) 734-5766
> Fax: (415) 355-0230
> E-mail:
> Mailing address:
> The J. David Gladstone Institutes
> 1650 Owens Street
> San Francisco, CA   94158
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