[Histonet] drosophila sectioning

[Rene J Buesa]

Anthony:
  Hennings (1900) described a method to soften chitin, but I don't recommend it (too many toxic components).
   
  Slifer and King (1933) developed another method:
  Dist. water (30 mL) + 95% Ethanol (80 mL) + phenol (4 mL), but Roonwall (1935) reduces the amount of phenol to 1-2 mL
   
  These phenol containing solutions are good to soften chitin and, once is soft, will be readily infiltrated with the paraffin. The insects should be fixed first.
  René J.
  

Anthony Gatt <Anthony.Gatt <@t> jefferson.edu> wrote:
  Thanks, Rene. Any advice on a softening agent and where it would fit into the protocol?

---- Original message ----
>Date: Fri, 11 May 2007 09:15:23 -0700 (PDT)
>From: Rene J Buesa 
>Subject: Re: [Histonet] (no subject) 
>To: Anthony.Gatt <@t> jefferson.edu, "histonet <@t> lists.utsouthwestern.edu" 
>
> Your problem resides in the fact that you have to
> "soften" the chitin before processing the heads,
> otherwise the chitin cannot be infiltrated by the
> paraffin, causing the tears you are having.
> Soften the chitin first and process afterwards.
> René J.
>
> Anthony Gatt wrote:
>
> Hello, I am currently sectioning drosophila heads
> and am tearing up the paraffin. Without the heads,
> I get beautiful ribbons so I suspect it is the
> sample itself. I am using a tissue processor with
> the following protocol after an o/n fix in 4% PFA.
>
> 15m -alcoholic formalin (x2)
> 30m -95% EtOH (x2)
> 30m -100% EtOH (x2)
> 30m -Histoclear (x3)
> 30m -Paraffin (x2)
> 45m -Paraffin
>
> Embed immediately in paraffin, cold block 30m,
> section next day.
>
> I am new to this an would greatly appreciate any
> help.
>
> Thank you,
>
> Anthony
>
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