Re: [Histonet] Help in brain tisuue processing

From:Geoff McAuliffe

Greetings Leila

If you are fixing the whole brain by immersion the interior morphology 
won't be very good. I suggest cutting the brain into 1 cm slices and=20
fixing these for 1-2 weeks. You will be able to see enough of the=20
anatomy to know the order of the slices.
If you must process the brain whole you will need to fix by vascular=20
perfusion to see much (any) of the interior anatomy and your processing 
will have to be much longer.
The rest of your schedule does not allow enough time for dehydration,=20
especially on a whole brain. The tissue does not stick to the slides=20
because it was not properly dehydrated so the paraffin does not infiltrate.
On the other hand, all night in hot paraffin is too long and produces=20
brittle tissue.

I suggest the following for 1 cm slices, not a whole goat brain.
After fixation, wash in water for several hours or overnight.
50% ethanol 1-2 hours. Agitation of the tissue duringhis and subsequent 
steps is highly desirable.
70% ethanol 1-2 hours.
95% ethanol 1 hour.
100% ethanol 2 changes 1 hour each.
1:1 mix of 100% ethanol:xylene 1 hour.
Xylene, 2 changes 1 hour each.
3 changes of melted paraffin, under vacuum if possible, 45 min each.=20
Heat the wax only a few degrees above the melting point.

Leila Ramos wrote:

> Dear Mr. or Ms
> We are working in a small lab from Canarias Island (Spain). We work with
> goat brain with manual procedure, our problems are several in the
> processing:
>   1. We obtain very dry blocks, when we try to cut with the microtome,
>    the tissues are fragile and it is impossible to obtain the correct 
> tissue.
>   2. When "we get proper block" (we can cut better the tissue), then we
>   try to put in the float, the tissue appears like a white colour in the
>   slide. After when we finish the stain process (hematoxyline-eosina and
>   kluver barrera both of them) the tissues are not attached on the glass
>   slides.
> I explain you in fast way our protocol:
>   1. Fixation: the whole brain is embedded in Formol 10% (1 week
>   minimum)
>   2. Dehydration: 80º etanol 30 minutes; 96º 30 min x 2 times; 100 º 30
>   min x 5 times; xylene 1 hour x 2 times
>   3. Tissue embedding in paraffin. In cassetes with pure paraffin all
>   night- Only 1 step other times 2 changes in paraffin 1 hour
>   4. Cut with microtome at 8 um, slide with polylisine.
>   5. Stain process (Hematoxyline-Eosine and kluver barrera)
> Thank a lot!!
> Leila Ramos
> Instituto de Investigación y Ciencias de Puerto del Rosario
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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