Re: [Histonet] Manual Paraffin Embedding

From:Merced Leiker

Thanks for all the detail, Bryan!  We will try this and see how it works. 
Thanks to everyone else who replied as well, your tips are taken into 
consideration, too.


--On Tuesday, September 09, 2008 3:21 PM -0700 Bryan Llewellyn 

> I don't do this anymore, nor for 40 years now, but this is what we used
> to do aeons ago.
> 1. Fix in 10% NBF for 48 hours.
> 2 Rinse off excess with tap water for 1 minute.
> 3. Select pieces of tissue with maximum dimensions of 2cm x 1.5 cm x 0.3
> cm.
> 4. Place into cassettes if you have them, then into a large container.
> If you do not have cassettes, place into small jars, each case in a
> different jar.  Place a label in each cassette or each jar with the case
> ID.
> 5. Cover the tissue or cassettes with 70% ethanol, methylated spirits or
> isopropanol, agitate gently.  Leave overnight.
> 6. Next morning, replace the 70& alcohol with 85% alcohol, leave for the
> day, agitating gently periodically.
> 7. Before leaving in the evening, replace the alcohol with 95% alcohol,
> agitate gently and leave overnight.
> 8. Next morning, replace the alcohol with 100% alcohol, gently agitate
> periodically.  Repeat at noon and before you 9. leave for the evening,
> gently agitating.
> 10. Next morning, replace the alcohol with clearant, preferably xylene or
> toluene.  Leave for one hour, gently agitating periodically.
> 11. Repeat the clearant twice more, agitate gently.
> 12. Place into premelted paraffin wax for 1 hour at 65C.  Check
> periodically and when all congealed wax has remelted, agitate gently.
> 13. Repeat at least twice more, preferably under vacuum - not too strong.
> Some technologists used to leave the final change overnight.  Doing so
> doesn't do much harm and improves penetration.  Agitation can't be done
> under vacuum, so release the vacuum periodically, agitate and reapply it.
> 14. Block out into molds.  (If you do not have molds, use a metal -
> tinned steel or aluminum - lid with a depth of 1cm.  LIGHTLY coat it with
> glycerol first.)  Place a thin (3mm) layer of hot wax in the mold and
> place the tissue, with the surface to be sectioned down, into it.  Top up
> the mold with wax so there is at least 2-3 mm wax over the top of the
> tissue.  Put the ID label conspiciously next to the tissue.  Do NOT block
> out more than one tissue or case without placing the ID labels, this WILL
> lead to serious identification errors.  Do all this by the oven and keep
> the door closed as much as possible.  Work fast so that the wax around
> the tissues does not begin to congeal as that causes problems during
> sectioning and floating out. You must get the wax around the tissue and
> the wax in the mold to blend completely, so if the wax congeals around
> the tissue, leave it to remelt before blocking out.  Many of us used to
> keep a bunsen burner alight and flame the top of the molds to keep the
> wax molten - a practice probably considered unsafe now.
> 15. Allow the wax in the mold to skin over thoroughly, then GENTLY lower
> into cold tap water to cool.
> 16. When completely cold and solid, use a heavy knife to score and trim
> the wax blocks.
> 17. To section, melt the trimmed block onto the block holder.
> Bryan Llewellyn
> ----- Original Message ----- From: "Merced Leiker" 
> To: 
> Sent: Tuesday, September 09, 2008 1:54 PM
> Subject: [Histonet] Manual Paraffin Embedding
>> Does anyone process and embed tissues manually instead of using
>> automated  and expensive equipment?  Can you tell me how you do it?
>> Thanks.
>> Merced
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Merced M Leiker
Research Technician II
354 BRB (Lee Lab) / 140 Farber Hall (mail)
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

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