ImmPRESS Polymer Detection Following BaseScope In Situ Hybridization

Complete BaseScope in situ hybridization using 35um mouse brain sections. Do not dry slides.

For BaseScope protocol see : (dx.doi.org/10.17504/protocols.io.5qpvo364zv4o/v1)

Wash slides 2 times in PBS at room temp 5 minutes each wash

Block sections in 0.4% Bovine Serum Albumin (BSA), 10% horse serum, 0.3% triton X-100 for 1 hour at room temp

Incubate sections in rabbit anti TH (1:500) in 0.4% BSA, 5% horse serum and 0.1% tritonX-100 overnight at 4 degrees C

Wash slides in PBS at room temp (3 times 5 minutes each wash)

Incubate slides in secondary antibody (ImmPRESS-AP Polymer Reagent) at room temp for 2 hours.

Wash slides in PBS at room temp (3 times 5 minutes each wash)

Prepare the Alkaline Phosphatase Substrate

a. Make 2.5mL 100mM Tris-HCl, pH 8.2

b. Add 1 drop of Reagent 1

c. Add 1 drop of Reagent 2

d. Add 1 drop of Reagent 3

e. Mix well – use immediately

Incubate sections in alkaline phosphatase substrate for 5 minutes

Wash slides in PBS (2 times 5 min each wash)

Dry slides at 60°C for ≥15min until they are completely dry

Coverslip slides using VectaMount

Store slides at room temp