Re: Myelin processing

From:Bryan Hewlett

Xiangjun Chen,

I think the dictum that I was taught applies here.
"First we fix - and then we process"!

Perfusion for 10 min with 4% formaldehyde, followed by post fixation in the
same for 1-2 hours, doesn't even begin to approach adequate fixation for any
tissue, particularly CNS and PNS. Any slight immediate cross-linking, which
may have occurred, will be rapidly reversed by the 70% alcohol. Particularly
if the tissues are on a "DELAY" program.
Your tissues are really being fixed by the processing alcohols, not by
formaldehyde. I'm not surprised that the myelin structures are not well
preserved!
I would recommend that you first fix the tissue adequately, for a minimum of
24 hours, prior to processing.
I would also point out, that the adult mouse brain block thickness should be
no more than 2 mm with the process times you are using (d= K x the square
root of time, K for ETOH is 1.0). In addition, the use of vacuum for
dehydration is counter-productive. Diffusion is faster at higher pressures.

Bryan,

(Irascible old guy)




----- Original Message -----
From: 
To: 
Cc: 
Sent: Thursday, April 17, 2003 12:30 PM
Subject: RE: Myelin processing


> Hi,
>
> We usually intracardically perfuse the mice using 4% paraformadehyde(rinse
> blood vessels by saline before that) about 10 min, then postfix 1-2 hrs in
> the same fix. The programs we use in Leica TP1020 are as follows:
>
> Adult mouse brain:
> STATION# Reagent Vaccum Time
> 1 70%EtOH off 25min
> 2 80%EtOH on 25min
> 3 95%EtOH on 25min
> 4 95%EtOH on 25min
> 5 100%EtOH on 25min
> 6 100%EtOH on 25min
> 7 100%EtOH on 25min
> 8 Xylene on 25min
> 9 Xylene on 25min
> 10 Xylene on 25min
> 11 Paraffin on 45min
> 12 Paraffin on 45min
>
> Small Large Nerve(rat nerves or mouse sciatic nerve)
> STATION# Reagent Vaccum Time
> 1 70%EtOH off 10min
> 2 80%EtOH on 10min
> 3 95%EtOH on 10min
> 4 95%EtOH on 10min
> 5 100%EtOH on 10min
> 6 100%EtOH on 10min
> 7 100%EtOH on 15min
> 8 Xylene on 15min
> 9 Xylene on 15min
> 10 Xylene on 15min
> 11 Paraffin on 30min
> 12 Paraffin on 30min
>
> Usually the specimens are hold until the "DELAY" program runs in the late
> night.Because this processor only has 2 stations for paraffin(previously
the
> same protocol which has three paraffin stations,30 min each, worked well
in
> other brand processor) we modified to 45 min each. We found some area in
> brain and spinal cord still have problem of keep the myelin structure. Any
> suggestions about this?
>
> Xiangjun Chen
> Department of Neurology
> University of Chicago
> xchen@neurology.bsd.uchicago.edu
>
> -----Original Message-----
> From: Pamela Marcum
> To: xchen@neurology.bsd.uchicago.edu
> Sent: 4/17/2003 7:43 AM
> Subject: RE: Myelin processing
>
> Good Morning,
> Could you please send or publish your current processing schedule with
> reagents, times, temperatures and vacuum?  This will help understand
> where
> the problem may be and why it is happening.  Just from your question I
> cannot do anything except hazard a guess and that is not a fair way to
> help.
> If you just put the program on HistoNet with you question we can move
> faster.  Thanks,  Pam Marcum
>
> > -----Original Message-----
> > From: xchen@neurology.bsd.uchicago.edu
> > [mailto:xchen@neurology.bsd.uchicago.edu]
> > Sent: Wednesday, April 16, 2003 9:14 PM
> > To: histonet@pathology.swmed.edu
> > Subject: Myelin processing
> >
> >
> > Does anybody there know of the paraffin tissue processing
> > protocol for mouse
> > CNS and PNS using Leica TP1020 automatic tissue processor? We are
> having a
> > hard time to preserve the myelin structure of mouse tissue. Any input
> is
> > highly appreciated.
> >
> > Xiangjun Chen
> > University of Chicago
> > xchen@neurology.bsd.uchicago.edu
> >
> >
>
>





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