[Histonet] Sections falling off slides

[Montina Van Meter]

Brian,
   I perfuse rat and mouse brains with 4% paraformaldehyde, post-fix for
~2 hours and then place tissue in 30% sucrose overnight.  I routinely
section the brains at 40-60 microns and mount them onto chrome-alum
subbed slides.  I allow the slides to air dry overnight and then place
them in the -80 C. freezer.  I don't have any problems with the tissue
falling off.  Using the plus slides should work the same way as long as
you let them dry overnight - 20 min. just isn't long enough. (I also
wipe away any excess moisture around the sections with kimwipes.)  You
will not get the same amount of stain penetration as free floating, but
it does save on chemicals.  Hope this helps...

Tina Van Meter 




Montina J. Van Meter
Lab Coordinator
Autonomic Neuroscience
Pennington Biomedical Research Center
Baton Rouge, LA  70808
225-603-0953
vanmetmj <@t> pbrc.edu

>>> "Patsy Ruegg" <pruegg <@t> ihctech.net> 11/13/04 1:30 PM >>>
Brian if you can cut thinner the sections will stay on slides better,
also
airdrying over night instead of just 20 minutes would help, if you could
fix
the sections in something with alcohol in it that might help too, we use
a
formalin/methanol/acetone mixture, this is a tuff order for sections as
thick as you are doing and brain is also hard to keep on the slide.  I
have
a fan that I leave difficult sections on over night to dry, the fan lays
flat and slides sit in a rack on their side and the fan blows air on the
slides.
Patsy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Brian
Dias
Sent: Saturday, November 06, 2004 8:13 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Sections falling off slides


hi
   i'm trying to optimize my immunohistochemistry protocol on slides and
am
runnning into a few problems.
1. i'd like to know if anyone has any strong opinions (either
positive/negative) about conducting such protocols on slides v/s
free-floating. i'd like to be able to do this on slides because mounting
my
sections after performing a free-floating technique is virutally
impossible
due to the fragile nature and small size of of my sections.

2. i run into the problem of my sectiions falling off the slide whilst
doing my ICC

    the brain is dissected form the skull post-perfusion with saline and
4%
paraformaldehyde
    it is then stored in 4% paraformaldehyde for 3 days at 4 C
    then transferred to 20%sucrose/1X PBS and stored at 4 C for 3 days
before sectioning on the cryostat at a 30-50um thickness and mounted on
Fisher Probe-On Superfrost Plus slides. slides are stored at -80C after
they've dried (approx. 20 mins).
    just before (the night before/2 hours before) performing the ICC, i
remove the slides from the freezer and allow to air-dry.
    immerse slides in 4% paraformaldehyde for 10 mins at RT in a dish
    1X PBS wash at RT in a dish
    then normal ICC steps by overlaying solution on slides and here's
where
sections come peeling off.

i'd appreciate any input on the matter

thanks a ton
regards
brian




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                                                     Brian George Dias

    Graduate Student (Crews lab)            Office Phone #: 512-475-6738
    The University of Texas at
Austin        E-mail:   brian.dias <@t> mail.utexas.edu
    Institute for Neuroscience                  Website:
<https://webspace.utexas.edu/bgd85/b.htm>https://webspace.utexas.edu/bgd85/b
.htm

    Patterson 56
    1 University Station Stop C0930
    Austin, TX 78712
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