FITC probe and washes for fluorescent in situ

Chema developed Tt vasa using FITC probe for regular and fluorescent in situ and also Tt nanos. He washed the fluorescent ones according to the protocol from (10.1186/2041-9139-2-6) (basically the 20 minutes wash with detergent), which consists of:

Fixed larvae hybridized with Digoxigenin-11-UTP (Roche Applied Science, Indiana- polis, IN, USA) labelled Tt-c-opsin and Fluorescein-12-UTP (Roche Applied Science, Indianapolis, IN, USA) labelled Tt-Pax6 probes at 2.5 ng/μl each, for 48 hours at 60°C. Larvae were incubated overnight with Anti-digoxigenin-POD antibody (Roche Applied Science, Indianapolis, IN, USA) at a 1:1000 dilution in 1× Blocking Reagent (Roche Applied Science, Indianapolis, IN, USA) overnight at 4°C, and stained with TSA Plus Cyanine 5 at a 1:100 dilution for 60 minutes at room temperature. Following staining, peroxidase activity was extinguished by incubation in 2.7% hydrogen peroxide in 1× PBS buffer for 90 minutes. Subsequently, larvae were incubated overnight with Anti-fluorescein-POD antibody (Roche Applied Science, Indianapolis, IN, USA) at a 1:1000 dilution in 1× Blocking Reagent (Roche Applied Science, Indianapolis, IN, USA) overnight at 4°C, and stained with TSA Plus Tetramethylrhodamine at a 1:100 dilution for 60 minutes at room temperature. Stained larvae were washed for 48 hours with multiple exchanges of 1× PBS buffer, and cleared with 80% glycerol.

The washes apparently reduced the background, though also the signal. I’ll check the samples in the confocal.

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