[Histonet] Sections falling off slides

[Brian Dias]

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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