[Histonet] autofluorescence

From:"Sasa Jovanovic"

Hi everyone
Please help! In my project I am using immunohostochemistry to determine
presence/absence of integrins in rat uterine tissue. I am trying to make it
work for several months now with both alpha V beta 3 (immunoflorescence) and
alpha 4 beta 1 integrin (using DAKO ARK kit) and use of monoclonal
antibodies. My biggest problem so far is staining in the negative control
(positive and negative control stain exactly the same) and auto florescence.
I am following the Santa Cruz Protocol for immuno-fluorescence staining of
frozen tissue. I snap freeze rat uterine tissue in liquid nitrogen, cut 6 um
thin sections and adhere them on silane pre-treated slides and dry
overnight...... then I fix them in cold acetone for 10 minutes and proceed
with staining as follows:
- wash slides in three changes of PBS (pH9)
-incubate slides for 5-10 minutes in 0.1 H2O2 in PBS to quench endogenous
peroxidase activity
- wash slides in PBS 2x5minutes
- incubate slides with 10% normal goat  blocking serum in PBS for 20 min to
suppress non - specific binding of Ig G
- wash in PBS (3x5min)
- incubate with primary antibody for 60 minutes
- wash in 3 changes of PBS
- incubate for 45 minutes with FITC conjugated secondary diluted in PBS with
1.5-3% normal blocking serum in dark chamber
- wash in 3 changes of PBS
- mount and coverslip directly from PBS with aqueous mounting medium
Both primary (sc-7312)and secondary (sc-2010) antibody were purchased from
I hope this is sufficient info to help you find some answers to my problem
Thank you in advance
Kind regards
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