Author Topic: Graded Ethanol Series  (Read 9681 times)

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Offline CUgrad001

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Graded Ethanol Series
« on: January 07, 2009, 06:24:04 PM »
Hi everyone, I am new to this site and so I hope I am posting in the correct place.  I am attempting to do a H2O replacement technique using a graded ethanol series (70%-->80%-->95%-->100%) on human soft tissue, intervertebral disc to be exact.  The tissue is approx 1cm X 1cm X 1cm (for subsequent SEM CO2 drying and SEM imaging) and my question is this.  How long do you all think I need to "soak" my tissue cube in each solution before moving onto the next?  The literature is rather confusing and doesn't appear to have a hard suggestion.  If it helps at all, it's going to be done at room temp.  Thanks!

Graded Ethanol Series
« on: January 07, 2009, 06:24:04 PM »

Offline pweston

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Re: Graded Ethanol Series
« Reply #1 on: February 16, 2009, 11:48:06 AM »
This is the protocol I use similar to that in TEM:

After rinsing several times with 0.1 M sodium cacodylate buffer,

2% osmium tetroxide in 0.1 M buffer...30 min

25% ethanol...10 min
50%...10 min
70%...10 min (can stay overnight in this step if you are pressed for time)
95%...2 x 10 min
100% (200 proof)  3 x 5 min

Then you can do your critical point drying with CO2.  When doing cells on coverslips I do 5 minute dehydrations and a 15 min osmium step.

Hope this helps.

Re: Graded Ethanol Series
« Reply #1 on: February 16, 2009, 11:48:06 AM »