IN SITU-PCR: looking for advice

[Anders Johannisson]

We are planning to start an in situ-PCR project, in order to detect low
copies of viral mRNA in brain tissue sections. The machine we use is the
new in situ-PCR thermocycler from Perkin-Elmer. We are very grateful for
all advice we can get on the subject, but particularly regarding the
following issues:

1) What type of section is the best one to start with: frozen or
paraffin-embedded? Is it really possible to maintain the morphology on
frozen sections?

2) Which detection system is the best alternative: hybridization with a
non-radioactive probe after the PCR reaction, or direct incorporation of
dig-dUTP in the PCR mix?

We would be most grateful for some advice from people experienced in this
technique. 

Best regards,

Anna-Lena Lundgren & Björn Rozell
Dept. of Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
E-mail address: Anna-Lena.Lundgren at pat.slu.se