[Histonet] My ICC Protocol- Sections getting chewed

[Brian Dias]

Protocol:
Fresh frozen
Animals decapped and brains frozen on dry ice.   Stored at 
-80C.   Sectioned (16um) sections on cryostat on to Superfrost Plus 
slides.  Stored at -80C.   On day of ICC, removed from -80C,   thawed for 
30 mins,   ice-cold (-20C) acetone (10mins),   air 
dried(30mins),   1XPBS-RT-5min(x2),   3%H2O2/1XPBS/0.3%TritonX-100 
RT-30mins,   1XPBS-RT-5min(x2),   Block (1XPBS/4%NGS/0.3%TritonX-100)-RT-1 
hr,   1 Ab 
(1XPBS/2%NGS/0.3%TritonX-100)-RT-O/N,   1XPBS-RT-10min(x2),   2Ab 
(1XPBS/2%NGS/0.3%TritonX-100)-RT-2hrs,   1XPBS-RT-10min(x2), ABC-RT- 
1hr,   1XPBS-RT-10min(x2),   DAB-RT-10mins,   1XPBS-RT-10min(x2),   air 
dry,   dehydrate and coverslip

Paraformaldehyde fixed
Animals decapped and heads in 4%PFA-4C-5 hrs,   Brains dissected, stored in 
4%PFA-4C-3 hrs,   Brains transferred to 20%sucrose/1XPBS-4C- approx. 12 
hours,   Sectioned (16um) sections on cryostat on to Superfrost Plus 
slides,   Allowed to dry at RT O/N,   Stored at -80C.   On day of ICC, 
removed from -80C, thawed for 30 
mins,   4%PFA-RT-10mins,    1XPBS-RT-5min(x2), 
3%H2O2/1XPBS/0.3%TritonX-100 RT-30mins,    1XPBS-RT-5min(x2),    1% 
NaBorohydride in 1XPBS-RT-20mins,    1XPBS-RT-5min(x2),    Block 
(1XPBS/4%NGS/0.3%TritonX-100)-RT-1 hr,    1 Ab 
(1XPBS/2%NGS/0.3%TritonX-100)-RT-O/N,    1XPBS-RT-10min(x2),    2Ab 
(1XPBS/2%NGS/0.3%TritonX-100)-RT-2hrs, 1XPBS-RT-10min(x2),    ABC-RT- 
1hr,    1XPBS-RT-10min(x2),    DAB-RT-10mins,    1XPBS-RT-10min(x2),    air 
dry,   dehydrate and coverslip


hi everyone,
    i'm trying to get my immunohistochemistry on slides to work for several 
antigens and have been successful in doing so in the past whilst preserving 
tissue morphology on the slides at the same time. of late, i've been 
getting tissue that looks chewed up, pock-marked (really badly)after i get 
thru' the ICC (this happens to both my fresh frozen as well as perfused 
section slides, more with the fresh frozen). the antigen staining still 
exists BUT is extremely hazy due to the tissue morphology.
    any input is welcome.
thanks a ton
regards
b

ps just thought this might be of interest to the list users: in the past i 
had issues with my 30um sections falling off slides whilst performing my 
ICC. that problem was solved by using Superfrost Plus Gold slides from Erie 
Scientific (thanks barbara!!!)

____________________________________________________________________________
    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
____________________________________________________________________________