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Author Topic: unfixed tissue at room temperture  (Read 14114 times)

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Offline pweston

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unfixed tissue at room temperture
« on: October 16, 2008, 12:21:44 PM »
I see people cryosectioning flash frozen, unfixed tissue, and leaving the sections on slides at room temperture for many minutes up to over an hour.  This seems dangerous to me, in that the unfixed tissue proteins can begin to decompose thereby affecting any immunohistochemical labeling.  I always keep my newly sectioned tissue/slides in the cryostat as I am sectioning, store at -20 degrees C , and fix the sections right out of the freezer in cold acetone.  Is my way overkill, or are these other people doing something wrong.  I realize that fixed tissue is another matter and precautions such as mine are not necessary for this type of sample.

Thanks for your input.

unfixed tissue at room temperture
« on: October 16, 2008, 12:21:44 PM »

Offline richard03

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Re: unfixed tissue at room temperture
« Reply #1 on: October 16, 2008, 11:10:37 PM »
I always air dry unfixed fresh frozen tissue slides for at least 30 minutes at room temp before cold acetone fixation and air dry again for another 30 minutes before immunostaining. Never had problems with staining quality.

Offline Cardio

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Re: unfixed tissue at room temperture
« Reply #2 on: November 10, 2008, 04:54:39 PM »
I use to think that this was a dangerous practice but it probably doesn't matter unless the protein is really unstable or in very low concentration.

I recently did an H and E on non-fixed slides stored at room temp for 3 years and no it wasn't my samples. I wouldn't recommend it but the beef jerky hearts stained.


Offline ImmunoNYC

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Re: unfixed tissue at room temperture
« Reply #3 on: December 10, 2008, 08:31:21 PM »
We leave our slides of unfixed frozwn sections out overnight at RT prior to staining or freezing. I have never had a problem. I worry with your technique about freeze-thawing and frost artifact (afterall the sections refreeze when you put in cryostat and then might thaw a bit before going in freezer) as well as section adherance. I am interested to hear your experiences with this.

Offline CanuckPhD

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Re: unfixed tissue at room temperture
« Reply #4 on: December 11, 2008, 01:24:45 AM »
I know some people that follow Paula's protocol when they are wanting to stain cytokines in EAE brain tissue, as they say that the cytokines will be degraded during drying out. They always had decent staining but the morphology wasn't great. For them the trade off was worth it.

Offline MT Scientist

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Offline ImmunoNYC

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Re: unfixed tissue at room temperture
« Reply #6 on: January 19, 2009, 04:03:11 PM »
So let me understand, the tissue was melted onto the slide and immediately put back in cryostat to freeze again?

Offline pweston

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Re: unfixed tissue at room temperture
« Reply #7 on: January 29, 2009, 12:23:55 PM »
Yes, that is correct. 

Re: unfixed tissue at room temperture
« Reply #7 on: January 29, 2009, 12:23:55 PM »