Basic histology:getting the specimin to stick!

From:Stephanie Moore <>

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.

Lab Mgr & Tech
Brandeis University

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