More details on my problem...

From:Bonnie Wayne <>

Here's my first post:

I am having some difficulties with my histology work.  I have rat cerebellum
and brainstem embedded in gelatin, sliced, and stained with cresyl violet.
Things are going fairly well except two things.

1) The gelatin likes to curl.  The brain section (30 um) sticks
wonderfully but the excess gelatin loves to lift off of the slide.  I
tried cutting the excess gelatin off but then the brainstem wants to lift
slightly.  Any suggestions on how to get the gelatin to stick?

2) The cerebellum has some cracking of the tissue.  Thought you might have
some advice that could help me prevent this.

If I need to give you more details on the staining/subbing process, let me
know.  I really appreciate it.  - Bonnie
Here's a reply to my post:

The way it's usually used, gelatin does not infiltrate an object;
it surrounds it and enters the larger crevices. Its cutting and
properties and stength are enhanced by formaldehyde fixation of
the "embedded" and trimmed block. After 12-24 in the fixative
the gelatin is cross-linked enough to be insoluble. It also
won't melt when warmed, and you have nice square frozen sections
that are more easily manipulated than brain-shaped ones. You didn't
say if your gelatin blocks were fixed. You also didn't say how
long the brain was fixed for, or if a cryoprotectant (it's usually
surose) was used, either before or after putting in gelatin. It
makes sense to do the cryoprotection last, in the hope of getting
the most even consistency of tissue-plus-gelatin.

Section adhesion is also affected by the way you mount the sections
and by any pre-treatments that you apply to the slides (cleaning,
adhesives etc). For obvious reasons mounting from water is preferable
to mounting from a solution containing dissolved salts that may end
up as insoluble crystals under the sections.

John A. Kiernan

Here's my reply:

Yes, the gelatin blocks are fixed in formaldehyde as John described.
They are stored in the formaldehyde until used.  This could range from 48
hrs to several weeks. The blocks are fixed but not frozen.  I've never
heard of a cryoprotectant but assume that it is an agent to protect the
tissue from damage due to freezing.  As I am not freezing anything, I
don't believe it's necessary to use a cryoprotectant.  (Pardon my ignorance,
laugh now, but help me later :P )  I know that the slides were subbed
right out of the box using biobond.  The subbed slides are stored in the box.
During mounting, each section is place in distilled H20 and mounted on the
slide underwater.  I originally tried drying them on the slide warmer but
could see the gelatin lifting as the water evaporated.  I've tried drying them
 outside the slide warmer both propped up and laid flat.  None of these
attempts have fixed the fact that the gelatin lifts off the slide.  The
brain stays wonderfully flat.  We noticed that if you hold a slide up to your
mouth, and breathe on it, the gelatin lays instantly flat again.  Of course,
this observation doesn't help me when I am putting the slides through a
series of alcohol baths.  I think I am looking for a way to sub the slides
so that the gelatin will be as eager to stick as the tissue is.  Or
perhaps, I need to add something to the gelatin to make it more
susceptible to sticking to glass.  *sigh*
Another comment about my first cry for help:

*Gelatin temp. should not be over 60C (I use 58C) or the gelatin will
become denatured.

Alright maybe this is my problem.  Question, how would I know if the
gelatin is denatured?  This is how I prepare the gelatin:  I boil some
distilled H20, I take it off the burner and dump the proper amount of
gelatin in, stirring like crazy to break up all the gelatin and get the
mixture all uniform, then I poor it onto my rat brains in molds and let
cool in the fridge.  So, probably, the gelatin is greater than 60 degree
C.  It seems just fine.  If it were denatured, would I be able to tell?
Would the denaturation of the gelatin explain why it won't stick the

Thanks so much, Bonnie J. Wayne (receiving a B.S. in Mathematics and
Psychology with a minor in Biology this Saturday.  Woo Hoo!)

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