[Histonet] Mounting Gelatine Embedded Cryostat CNS Sections

[Maria Mejia]

Dear Gerald,

For large brain specimens, I always embed them in gelatin (my recipe) and to
section them I use the sliding microtome and section 30um to 50ums.
These sections are free-floated in 0.1M phosphate buffer and are mounted on
gelatin sub slides. As for in-house sub slides there are numerous recipes
and each differs slightly in each lab.

To gel sub slides use UNCOATED glass slides and clean...again (even though
it says on the box - clean slides). I place the slides in a chromerge acid
solution overnight (using hood).  I can provide this recipe if you are 
interested.
Next day, the slides are removed from the acid solution and washed in 
running
tap water for 1-2 hours with final rinse in distilled water. Then I 
gelatin coat
using Sigma's 300 bloom gelatin.

Gelatin Subbing Solution
distilled H20 - (cold) - 500ml
gelatin (300 bloom) - 6gms
Stir to mix well with heat (58 degrees C - don't heat solution above 58C 
or it
will denature the gelatin.
Add chromium potassium sulfate (chrom alum) - 0.2gms and mix again.
Cool abit and filter using #4 paper.

When coating slides make sure the solution is NOT cool - makes for 
better even
coating. Air dry slides well and store in slide boxes. I keep these gel 
sub slide
for no longer than 6 months, then use a freshly made batch.

Yours

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185



germckeon <@t> excite.com wrote:

>
>Hello,
>
>I am currently working on spinal cord and am having trouble with sections floating off. I have embedded with gelatine, am performing cryostat sectioning and will be doing some of the more structural stains eg Kultschitsky's stain for myelin. I have been using Superfrost slides.
>
>Can anyone advise me regarding choices of slides, coatings, methodologies, etc which would maximise adherence?
>
>
>
>In gratitude for any advice received,
>
>Gerald McKeon
>
>
>
>
>
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