[Histonet] Why do some paraffin sections of brain wrinkle on thewater bath?

Fri Jun 3 14:11:49 CDT 2005

Hello Clarissa, I have some experience with human brain, and what you describe
is what I've noticed routinely for several years. That's just what brain does.

Thicker paraffin sections (10-15um) have more wavy puckering problems than do
thin sections (5um). Larger sections also have more problems. I cut autopsy
tissues up to 1x1.5in and giant sections up to 4x6in. They often have variable
puckering which seems related to white vs grey matter patterns in the tissue.
Hotter water bath helps increase the flattening (47C), but also puts you at
greater risk for exploding sections if the processing hasn't been perfect. The
tissue has shrunk quite a bit during routine processing and I imagine the hot
water bath makes it want to swell up to it's original size -- or more. The
puckering is the result of differential swelling and the restrictions from the
surrounding paraffin and other tissues. At 8um the paraffin border of the
section can resist the heat of the water bath and the pressure of the expanding
tissue, and prevent the tissue from spreading. Thin sections left on the water
bath tend to end up with irregular outlines, somewhat reflecting the tissue
shape and the placement of initial puckers. Blocks that have a wide margin
constrict the tissue more than those with narrow margins. I have trimmed the
margins from some sections on the bath and this allowed more even spreading. But
of course excessive spreading will show artifacts microscopically. You could try
thinner sections 7,6, etc until you find a new balance between signal strength
and puckering problems. Or warmer water in the bath. I have experimented with
floating sections on ethanols from 60-80% but wouldn't recommend it for routine
work. There's both the fire hazard of warmed alcohols and alot of handling
differences from normal water bath routines. However, it was impressive to see a
large section (2x3in.) floating flat with zero swell and minimal spread -- not
even collagen took up water from the ethanol bath. Along similar lines, you
could also experiment with turning the water bath temp down -- alot. Cooler
water won't swell the tissues as much. But you also have to have very good
microtomy technique since you can't rely on using the water bath to fix the
problems in the sections. It is possible to cut serial perfect sections of brain
-- no chatter, no compression, no wrinkling in the tissue -- but it takes more
effort, and more fresh blades. 

Puckers on the water bath don't necessarily have to translate into wrinkles on
the slides. Several things seem to influence whether the puckers become wrinkles
or simply go away with drying. Lots of my sections retain some gentle "waviness"
when I pick them up, but once stained, the waves are gone. Differential swelling
gives way to differential drying. In my experience, the exact things I do to dry
the slides can make a difference between having flat, wavy, or lifting sections
where originally there were puckers. Vertical draining at room temp for short
time (< 30min) followed by overnight in a 37C oven is what works best for me.
You could simply try varying your drying protocol. An old trick I was taught
here helps sometimes, especially if there are large puckers. You wet a filter
paper with 95-100% ETOH and place it on a flat surface. When you pick up the
section, briefly let it drain, then lay it down on the filter paper section side
down. Rub the back of the slide with firm pressure -- too much will make the
section stick to the paper. Separating the slide from the filter paper generally
goes smoothly -- surprising but true -- but takes a little practice. What you
end up with is a section that has been both physically flattened against the
slide and dehydrated (shrunk) at the same time. This is a technique for problem
sections, not routine use (although for giant sections the time is worth the
results). This trick doesn't get rid of real wrinkles, but it will tend to make
them stick to the slide instead of falling off. It also will instantly turn
large puckers into wrinkles -- squished.

I hope some of this rambling helps,
Neuropathology Lab
Brigham & Women's Hospital

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of CM Bush
Sent: Friday, June 03, 2005 1:54 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Why do some paraffin sections of brain wrinkle on
thewater bath?

Dear Histonet,
Seeking some some basic info:
Sometimes, when cutting 8 um sections of brain, mouse or human (in standard size
cassettes), the section will either wrinkle or pucker once on the water bath
(set at ~43C), and then other sections (from different blocks) are great and
flatten out, very well, looking pretty much like the tissue did at the face of
the block. 
The mouse brains seem to wrinkle more randomly, where human brain sections
pucker in a more regular pattern-does this depend at all on how the piece of
human brain was cut-in as far as which section of brain, or ratio of white
matter to gray, contours of the brain, or dehydration issues, or..?  
For the mouse brains, some extra time on the bath seems to iron out the wrinkles
pretty much.  Same for the human brain but not completely.  If I could draw a
picture, once the section of human brain has dried on to the slide there are
regular patterns of slight folds where the puckers where, arranged around the, I
think, tracks of white matter.
Like I said, sometimes these wrinkle and puckers take care of them selves while
on the water bath or drying on the slide- I'd like to try to improve the
sections, what I can do to fix this, or at least find out what is going on with
this, just to know.
Thank you so much for the help.

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