Dear Stephanie,

We regularly do Nissl and CO on rat brain sections prepared in the same way

>Brain block in OCT medium to be sliced on cryostat.  On someone's
>suggestion, I thaw-mounted the specimens onto gelatin-chrom-alum subbed
slides. Then I just air dry them in a clean environment (fume cabinet 
or covered on the bench) overnight at room temperature  and proceed 
with the staining the next morning.

We get good CO staining, and the sections rarely lift. The tissue has 
good preservation of the cells
and doesn't appear to be at all damaged.

If you want to store them after air drying, they can either go into a 
-80 freezer or a regular freezer in a glycerol solution. The trick is 
not to not to freeze them wet, as this makes crystals and damages the 
tissue when they thaw. Either keep them frozen during the entire 
exercise or dry them first.

Hope this helps, Cathy

---------------------------------------------------
Cathy Gorrie
Scientific Officer
Neural Injury Research Unit,
School of Anatomy,
University of New South Wales
Sydney, N.S.W. 2052

Phone: 61 2 9385 2462
Fax  : 61 2 9313 6252
e-mail: c.gorrie@unsw.edu.au


At 4:26 PM -0500 16/1/01, Stephanie Moore wrote:
>Okay Folks,
>
>Here is the story of what I did and in your vast cumulative knowledge, I
>am sure you will have no problem finding the flaws:
>
>Brain block in OCT medium to be sliced on cryostat.  On someone's
>suggestion, I thaw-mounted the specimens onto gelatin-chrom-alum subbed
>slides (had cold slides in cryostat chamber, put slice on and then put
>slide on my arm to "melt" the brain onto the slide).  Then put slides
>directly into a container sitting in dry ice to keep them cold.  Stored
>slices at -80 degrees C (reference had -65, but we don't have access to
>such a warm freezer :)  ).  First set of staining I did was Nissl and
>after removing the slides from the freezer and letting them sit at room
>temp for 20 minutes, I put them in a 45 deg. C oven (while references
>say overnight, I only had time to do it for a couple of hours...I
>figured it was for drying the slides and since I was going to dehydrate
>them anyway...).  That was all fine, Nissl went fine except for the
>edges of the slices:  they didn't keep sticking so they curled a little
>during processing.  Of course that is the part we need: the cortex.
>this was practice and I figured I would do things right next time around
>(overnight at 45).  Today I was taking additional practice slices from
>the -80 freezer and performing cytochrome oxidase staining.  My
>protocols say nothing about drying in the oven first, so I just let them
>sit at room temp for about 5 minutes and then continued onto my .1M
>phosphate buffer wash.  Well I guess you might know what happened:  my
>slices came right off of my treated slides.
>
>Now I am starting to believe that the "drying" is really a process for
>slices to become truly stuck.  Most of my experience is
>immunohistochemistry and immunofluorescence with things that do not need
>dehydration, clearing, and mounting.  I just mount and use an aqueous
>medium (Fluoromount (tm)).  I have traditionally had bad luck with the
>dehydration process because my slices keep falling off.  But I always
>neglected the drying in oven step because I was convince that it would
>ruin my slices (curl, crack, etc).  My lab doesn't have a slide warmer,
>but I do have a little oven I can use.  I have little formal histology
>training (mostly self-taught) but unfortunately I have more knowledge
>than anyone else in the lab!  I have read books, etc...but nothing like
>hands on or at least having expert suggestions from the pro's.
>
>Thanks,
>Stephanie
>Lab Mgr & Tech
>Brandeis University