[Histonet] Manual Paraffin Embedding

[Merced Leiker]

Bryan,

Just one question on applying the vacuum: Do you keep the melted paraffin 
at 65 while applying it, or slightly higher to make sure the wax doesn't 
solidify on the surface?

Thanks,
Merced

--On Wednesday, September 10, 2008 3:05 PM -0400 Merced Leiker 
<leiker <@t> buffalo.edu> wrote:

> 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.
>
> Merced
>
> --On Tuesday, September 09, 2008 3:21 PM -0700 Bryan Llewellyn
> <llewllew <@t> shaw.ca> wrote:
>
>> 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" <leiker <@t> buffalo.edu>
>> To: <histonet <@t> lists.utsouthwestern.edu>
>> 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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>>
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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
>
> In order to put yourself in someone else's shoes,
> you must first take off yours.
>
>
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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

In order to put yourself in someone else's shoes,
you must first take off yours.