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Author Topic: acetone fixation  (Read 33040 times)

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Offline apple

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acetone fixation
« on: January 30, 2006, 12:23:34 PM »
Hi,

Many people use "cold acetone" to fix cytospin slides. What's the exact temperature of the acetone? 4C or -20C? Will it make big difference?

Many thanks

acetone fixation
« on: January 30, 2006, 12:23:34 PM »

Offline ImmunoNYC

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acetone fixation
« Reply #1 on: January 30, 2006, 10:54:17 PM »
I fix my slides in room temperature acetone and its just fine. I never use cold or -20 deg acetone, I find there is no difference but I am curious to hear other people's opinions.[/color]

Offline richard03

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acetone fixation
« Reply #2 on: January 31, 2006, 12:04:45 AM »
Some people fix frozen sections immediately after sectioning and mounting on slides, or they fix the slide when it is still cold in freezer. In these cases, one should use cold acetone to prevent artifact from forming.

Another concern is that cold acetone may preserve antigen/enzyme better for IHC purpose than RT acetone.

I always use cold acetone (4 C) to fix frozen sections BUT have never compared the differences between RT, 4 C and -20 C. I may give it a try when my time is allowed and let you know the difference in terms of morphology and immunoreactivity.

Richard

Offline discman

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acetone fixation
« Reply #3 on: January 31, 2006, 06:14:59 AM »
For cytospins I always used -20C acetone and got very nice results with various antibodies. However, I did never use RT or 4C acetone, so I can't tell you if the temp really makes a difference.

GŁnther

Offline apple

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acetone fixation
« Reply #4 on: January 31, 2006, 05:48:32 PM »
Hi discman,

How about your cytospins? Do you fix them immeidately after centrifuge or fix them after taking out of freezer or -80C?
I made my cytospins and store at -80C. When I do ICC, I will take cytospins out from -80C, then leave at RT for 10 min. Then fix in -20C acetone.

Offline discman

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acetone fixation
« Reply #5 on: February 01, 2006, 02:39:46 AM »
Hi apple,

I usually dried the cytospins for at least an hour after centrifugation and fixed them then before storing them. That's what worked best for me.

GŁnther

Offline apple

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acetone fixation
« Reply #6 on: February 01, 2006, 01:00:41 PM »
Hi discman,

I do the same. I fix my cytospin after centrifugation (leave for air dry 2hr to overnight) in cold acetone for 1-2 min, then store at -80C. Before ICC staining, I will fix them again in cold acetone (-20C) for 10min. My results are OK. But comparing to 4% PFA fixation, I found 4% PFA produce much better fixation. However 4% PFA also induce autofluorescence.

Have you tried acetone/methanol (1:1) fixation?

Thanks

Offline richard03

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acetone fixation
« Reply #7 on: February 01, 2006, 11:18:23 PM »
Acetone fixation - Better staining, Worse morphology
PFA/Formain fixation - Better morphology, Worse staining.

Same as

Frozen section - Better staining, Worse morphology
Paraffin section - Better morphology, Worse staining (it is not always true).

Quote from: "apple"
Hi discman,

I do the same. I fix my cytospin after centrifugation (leave for air dry 2hr to overnight) in cold acetone for 1-2 min, then store at -80C. Before ICC staining, I will fix them again in cold acetone (-20C) for 10min. My results are OK. But comparing to 4% PFA fixation, I found 4% PFA produce much better fixation. However 4% PFA also induce autofluorescence.

Have you tried acetone/methanol (1:1) fixation?

Thanks

Offline apple

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acetone fixation
« Reply #8 on: February 02, 2006, 05:13:34 AM »
Thanks. Richard03.

Offline discman

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acetone fixation
« Reply #9 on: February 02, 2006, 08:23:55 AM »
Quote
Have you tried acetone/methanol (1:1) fixation?


Yes, I have tried this quite some time ago. If I remember correctly, I didn't see any noticable differences to acetone alone. This was when I had problems with acetone fixation on frozen mouse tissue. With these samples methanol/acetone didn't improve the problem, so I didn't follow this method further.

Once I tried cold methanol alone which didn't work too bad. I used it when I tried to stain cells directly grown on transwell inserts with an antibody that didn't like formalin/PFA. Got some nice stainings that way.

GŁnther

Offline apple

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acetone fixation
« Reply #10 on: February 02, 2006, 07:22:55 PM »
hi discman, how did you know that ab didn't like PFA? Did you try with PFA fixation before?

I also tried cold acetone/methanol fixation. I couldn't tell the difference with cold acetone alone.

Offline discman

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acetone fixation
« Reply #11 on: February 03, 2006, 05:04:36 AM »
Quote from: "apple"
hi discman, how did you know that ab didn't like PFA? Did you try with PFA fixation before?


No, I didn't try. I (naively  :?  ) trusted the manufacturer's recommendation.

acetone fixation
« Reply #11 on: February 03, 2006, 05:04:36 AM »