[Histonet] Re:Frozen Spleen w/bubbles

From:Jo Dee Fish

>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?
>How thick are the sections?
>What temperature are you sectioning at?  i.e. knife temperature? 
>Block/tissue temperature?
>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: jfish@gladstone.ucsf.edu

Mailing address:
The J. David Gladstone Institutes
1650 Owens Street
San Francisco, CA   94158

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