Propidium Iodide counterstaining

This nuclear stain also binds to cytoplasmic RNA, so the sections are pretreated with RNase A.

RNase A will be used at 500mg/mL of 0.1M PB; calculate 100-200ml per slide

Propidium iodide will be used at 5mg/mL 0.1M PB; calculate 100-200ml per slide

Process tissue as normal with primary and secondary antibodies

PI is not membrane permeable; if an antigen retrieval method was not used (i.e. trypsin digest), will need to perform briefly prior to the following steps

Preboil RNase A (to inactivate any residual DNase) for five minutes

Quench briefly on ice

Apply RNase A to slides, coverslip with parafilm, and let incubate at 35°C for 30 minutes

Rinse briefly with 0.1M PB

Apply propidium iodide and coverslip with parafilm; let incubate at room temperature for 1-5 minutes

Wash slides in 0.1M PB 2X5 minutes

Coverslip with fluormount and store in the dark