Re: [Histonet] Manual Paraffin Embedding

From:"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" 
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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