Basic Protocol 5).
Tyramide Signal Amplification (TSA) detection of digoxigenin-labeled probes
This alternative procedure for digoxigenin detection uses peroxidase-conjugated anibody in an amplification scheme involving biotinyl tyramide and subsequent detection with streptavidin-conjugated fluorochromes, peroxidase or alkaline phosphatase. This approach produces an increased signal as compared to Basic Protocol 4 and also enables the researcher to develop the signal as a fluorescent product compatible with a second non-radioactive hybridization histochemical or an immunohistochemical labeling.
(see also the protocols for Immunohistochemistry)
Materials
1. Transfer slides from SSPE wash above to Buffer 1 for two 5-min washes.
2. Transfer slides to Buffer 1 with 5% NGS and 0.6% Triton X-100 for 30 min.
3. Transfer slides to Buffer 1 with 5% NGS, 0.6% Triton X-100, and 1:300-1:600 anti-digoxigenin-POD for 2 hrs with gentle rocking, and then overnight at 4° C.
4. Transfer slides to Buffer 1 for three 5-min washes.
5. Transfer slides to 1x Diluent for 5 min.
6. Transfer slides to 1x Diluent with 1:50-1:100 Biotinyl tyramide for 10 min.
7. Wash slides with three 5-min washes in Buffer 1
Detection of the labeled probe may now proceed in several ways, through the use of streptavidin-Texas red or streptavidin-fluorescein (a), streptavidin-HRP (b) or streptavidin-alkaline phosphatase conjugates (c) in increasing order of sensitivity:
8a. Prepare a 1:2000 dilution of streptavidin-Texas red or of streptavidin-fluorescein in Buffer 1. Incubate slides in this solution for 60 min at room temperature.
9a. Wash slides four times, each time by immersing 5 min in fresh Buffer 1.
8b. Prepare a 1:3000 dilution of streptavidin-HRP conjugate in Buffer 1 containing 0.5% DuPont blocking reagent. Incubate slides in this solution for 60 min at room temperature.
9b. Wash slides four times, each time by immersing 5 min in fresh Buffer 2.
10b. Dissolve one DAB tablet and one urea/hydrogen peroxide tablet in 15 ml of Buffer 2. Transfer the slides to this solution and incubate until a signal develops at about 5-10 min.
Sometimes the signal becomes apparent while looking directly at the tissue section. With some other mRNAs, the sections need to be examined microscopically.
11b. Wash the slides twice with Buffer 2 using the technique of step 9b.
8c. Prepare a 1:25,000 dilution of streptavidin-alkaline phosphatase conjugate in Buffer 1 with 5% NGS, and 0.6% Triton X-100. Incubate slides in this solution for 60 min at room temperature. Then follow steps 4-9 of Basic Protocol 4.
12. Dip slides briefly into water and blow dry. Thoroughly dry slides on a slide warmer.
Slides should be thoroughly dried on the slide warmer before mounting of coverslips; otherwise the signal may be lost.
13. Apply coverslips to slides using Cytoseal 60 or similar organic-based mounting medium if not proceeding to autoradiographic detection (Basic Protocol 5).
One can also use fluorescein-12-UTP (Boehringer Mannheim) instead of digoxigenin-labeled UTP at the same concentrations to label RNA for nonradioactive hybridization histochemistry (Support Protocol 2 and Basic Protocol 3). The tyramide signal amplification (TSA; Alternate Protocol 1) method is then used with a HRPO-conjugated sheep polyclonal anti-fluorescein antibody (Boehringer Mannheim), followed by biotinylated-tyramide and streptavidin-HRP or streptavidin-alkaline phosphatase. The biotinylated-tyramide and streptavidin-alkaline phosphatase detection is very sensitive, close to that of radiolabeled probes. Furthermore, this permits the simulataneous detection of two different transcripts by non-radioactive means. After detection of the digxigenin-labeled probe (Basic Protocol 4), proceed without drying to Alternate Protocol 1 and use the HRPO-conjugated sheep polyclonal anti-fluorescein antibody in step 2, followed by biotinylated-tyramide and streptavidin-fluorochrome or streptavidin-HRP.
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