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Microtomy school

 
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Olympusman



Joined: 15 Jan 2012
Posts: 3863

PostPosted: Fri Apr 13, 2018 8:33 am    Post subject: Microtomy school Reply with quote

I had the opportunity the other day to visit the histology department at a medical center to learn what I could about microtomy. Absolutely fascinating. Mainly I learned a great deal about the importance of temperature at various stages of tissue preparation and embedding. Got to watch a cryomicrotome in action. Also learned about dehydrating tissue samples. Instead of progressively stronger steps in alcohol the technique they use is
10% buffered Formalin
70% Formalin 30% alcohol
Pure alcohol
Pure alcohol
Pure alcohol
Xylene

I also learned about prepared ionized slides which are used with silver stained tissues (brain, bone). Silver stained tissues don't adhere to normal slide and the tissue washes off during staining.
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Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA
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ChrisR
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Joined: 14 Mar 2009
Posts: 7956
Location: Near London, UK

PostPosted: Fri Apr 13, 2018 2:15 pm    Post subject: Reply with quote

I learned enough about it to be fascinated and, inevitably, accumulate some hardware. I doubt I have enough life left to actually get round to practicing, though!
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Olympusman



Joined: 15 Jan 2012
Posts: 3863

PostPosted: Sat Apr 14, 2018 1:09 pm    Post subject: Microtomy Reply with quote

Got out my binder of notes I accumulated over the years to review technique.
Purchased 10% buffered formalin and Potassium hydroxide. Also purchased a digital kitchen thermometer to establish heating of Paraplast and water bath atop a gravy warmer and a coffee cup warmer.
This is getting complicated for a hobby microscopist.
More hardware with a fixed amount of counter space in the lab.

Mike
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ChrisR
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Location: Near London, UK

PostPosted: Sun Apr 15, 2018 2:45 pm    Post subject: Reply with quote

It's also expensive. Minimum qty seems to be about $100.

I saw a Leitz microtome and bought it, expecting imagination to be fired. Still waiting.
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Olympusman



Joined: 15 Jan 2012
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PostPosted: Mon Apr 16, 2018 2:01 pm    Post subject: Buffered formalin Reply with quote

The histology lab I visited had a 2.5 gallon cubitainer of formalin. I purchased two pair of jars that appear to hold about 40 or 50 ml on Amazon from TissuePro Technology www.tissueprotech.com

As for the KOH, I now have a lifetime supply. I found it is very effective at dissolving residue accumulated in beakers when I decapsulate microchips.

Mike
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discomorphella



Joined: 01 Oct 2006
Posts: 602
Location: NW USA

PostPosted: Sat Apr 21, 2018 9:05 am    Post subject: Reply with quote

Hi Mike,

One thing you may want to invest in is a small crock pot and a simple proportional temperature controller. You can find used YSI models on ebay. That makes a really nice paraffin oven, and avoids the problem of getting water into your paraffin. Also, a gallon of Histoclear2 is a much better option than xylene, its nontoxic, and you can use it with regular 99% usp grade isopropyl alcohol, also comparatively nontoxic, and cheaply available. A very good dehydrating/embedding schedule that works for all manner of samples is
wash in tap water to remove formalin
50% IPA
70% IPA
90% IPA
95%
99%
99%
1:1 IPA / HC2
HC2
HC2
HC2 saturated with paraplast
PARA1
PARA2
PARA3
embed

This works very well for all tissues, botanical as well as animal histological samples. More rugged samples can skip the 50% IPA step and the 90% IPA step and the IPA/HC2 and HC2/para as well.
The main advantage for this technic is enhanced safety for you. Xylene is toxic, and can contain ethyl benzene which is worse. HC2 is far safer. You can run this whole protocol on your benchtop without a hood. Just get yourself some 120 ml jars (or use old spice jars) and a few tissue cassettes or make some out of screen and you're good to go. Also, the celloidin coating technique is a great way to keep your sections from falling off, if regular albumin won't work. The silanized (typically surface treated with APTES) are not as effective for long soaking in high pH solutions.
Get a good box of real, chemo-tested nitrile gloves, like Cardinal Health for example, and a good pair of safety glasses.

David
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Mike



Joined: 04 Aug 2006
Posts: 86
Location: Northeast Ohio

PostPosted: Thu May 17, 2018 7:13 am    Post subject: Reply with quote

Interesting topic as I have just started on my efforts to do thin sections, starting with botanical specimens. Since retirement, I am now aware of the benefit I enjoyed previously having labs available to me with their resources and ability to order most materials that are not so easily available to individuals.

As an alternative to xylene, I understand Histoclear I and II are good products; a little snooping on the MSDS shows HC I is D-limonene, a cyclic monoterpene derived from citrus products, and available widely including on evilBay. It is environmentally safe with easy disposal and the pure forms are suitable for human consumption.

HCII is primarily a (70% - 90%) aliphatic hydrocarbon, a Low boiling point hydrogen treated naphtha (CAS 64742-48-9) with the balance being D-limonene. The advantage claimed for HCII is 'greatly reduced orange odor' BUT it also is incompatible with many synthetic mounting media. I have a gallon of d-limonene and would certainly agree it has a 'distinct' orange odor, but at my use levels I actually find it quite pleasant.

Insofar as availability of d-limonene, I see multiple listings on evilBay and Amazon. Just a guess, but expect it is a seasonal item and pricing could very well drop significantly during the harvest of oranges.

As to 100% EtOH - about the only one available outside lab supply houses would be the venerable Everclear, 200 Proof, but one wonders of the necessity for absolute EtOH, recognizing its hydrophilic nature the sealed bottle when exposed to atmosphere will start to pick up water.

I have read the use of 99% IPA serves fine in place of EtOH, at least with botanical mounts, and will be proceeding with that method when I eventually get going. 99% IPA is readily available at drug stores, Wally World, Amazon, wherever. Just be sure you get the 99% as they will also have much lower concentrations available, I believe down to 70%. Some are using Kleen Strip denatured ROH which is a blend of EtOH (30 - 50%) and CH3OH (40 - 60%).

BTW - Paraplast Plus is regular Paraplast with a small percentage (>2%) of DMSO, I assume there to aid in tissue infiltration.

Just a few thoughts from someone hoping to do something with the microtome.

All the best,
Mike





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Mike



Joined: 04 Aug 2006
Posts: 86
Location: Northeast Ohio

PostPosted: Thu May 17, 2018 8:20 am    Post subject: Reply with quote

Hello David,

It's been some time!

Thanks for that listing of the steps you use - that will be a great starting point for me!

I assume your use of IPA in place of EtOH is from long experience as I recall you have been doing thin sections for years.

If I might pick your brain a bit - I have both an AO 320 as well as a Leitz microtome; also Tissue Tek Feather disposable blade holder so in good shape with equipment. Also have both Paraplast and Tissue Tek VIP embedding medium, so have that aspect covered. Do you have a ready source for the tissue cassettes? I had ordered some from Amazon but after over a month and no delivery in sight ended up cancelling the order.

How/when do you move the 'stages' downstream? E.g. - how long do you use the 50% IPA before it is exhausted, and then dilute the 70% to 50%, etc. or are all your baths made up fresh each time?

All the best,
Mike
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