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
--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
> 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
> 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"
> 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?
>> Histonet mailing list
> Histonet mailing list
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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you must first take off yours.
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