[Histonet] Help in brain tisuue processing

[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 
fixing these for 1-2 weeks. You will be able to see enough of the 
anatomy to know the order of the slices.
If you must process the brain whole you will need to fix by vascular 
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, 
especially on a whole brain. The tissue does not stick to the slides 
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 
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. 
Heat the wax only a few degrees above the melting point.
Embed


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
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>


-- 
--
**********************************************
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 
mcauliff <@t> umdnj.edu
**********************************************