Updated 7-27-22 by Patricia Graham
Single fluorescent in situ hybridization using TSA (Tyramide Signal Amplification) kit
See also the notes on the colorometric staining protocol.
Day 1 Hybridization (~ 3 hrs)
1. Remove MeOH, and rinse 2X with MeOH.
2. Remove MeOH, rock 2X in MeOH, 5 min each time.
3. Remove, and rock 1X in PBST/MeOH 1:1 for 5 min.
4. Remove, and add 4% PFA, Rock for 25 min.
Turn on the heat block and set to 95oC.
Make hybridization buffer (HB). You need 2.5 ml/sample for 500ul rinses and washes.
5. Remove, rock in PBST 4X5 min.
6. Remove most of the PBST, heat the tube at 95℃ for 5 min.
7. Remove, and rock in pre-warmed HB/PBST 1:1 for 10 min at 60oC.
8. Remove, and add pre-warmed HB (works OK with RT HB), and incubate at 60oC for 30 min. Periodically invert the tube to break up clumps of embryos (not actually necessary usually).
9. Repeat Step 8.
10. Heat the probes (~ 0.2 ul of the stock in 50 ul of HB. or 1 ul for a new probe) at 90-95℃ for 5 min. Transfer it to ice immediately.
11. Remove HB and add the probe (50 ul). Flick gently the tube a few times and incubate at 60℃ overnight.
a.Probe concentrations must be determined empirically. I have had different probes work at concentrations from 0.1 ng/ul to 25 ng/ul. Jie said that all of her probes worked at the same concentration in TSA as in colorometric, but this was not true for mine, and there seemed to be no consistent pattern as to whether you would need more or less concentrated probe for TSA.
b.Biotin is probably not your best choice for a label as there is a lot of endogenous biotin around that gives a high background. You can mitigate this somewhat by staining for a shorter time (see below), but it’s probably better to use dig, fluorescein or DNP.
c.Leave the HB for tomorrow on the bench.
Day 2 Post-hybridization wash and primary antibody labeling (~ 2 hrs)
12. Remove probe without losing any embryo, and add 500 ul HB, rinse 1X5 min at 60oC degrees.
13. Remove, and add HB, rock for 30 min at 60 degrees.
14. Repeat Step 13.
15. Remove, and add pre-warmed HB/PBST 1:1, Rock for 10 min at 60 degrees.
16. Remove, and rinse 1X in PBST, wash 3X5 min in PBST at 60 degrees.
17. Remove, and add 250 ul of antibody diluted in PBST (1:250 anti-fluorescein, rabbit IgG or 1:250 anti-dig, mouse IgG). Incubate O/N at 4 degrees.
Day 3 HRP-conjugated secondary antibody and tyramide labeling , fluorescence detection (~ 5 hrs)
18. Remove primary antibody, and rinse once with PBST.
19. Remove, wash with PBST 4X15 min.
20. Remove, and add 1:100 HRP conjugated secondary antibody (100ul/sample), RT 2 hrs.
21. Remove, rinse 1X with PBST and then wash 4x15 min with PBST.
22. Remove, add 1:100 tyramide in amplification buffer working solution (add 1 ul of 30% H2O2 from the kit into 200 ul of amplification buffer from the kit to get 0.15% H2O2, then add 1:100 this intermediate dilution to amplification buffer to make 0.0015% H2O2), rock at RT for 15 min. (Wrap the tube with foil to block light).
Note: the amplification buffer working solution has to be made fresh before each use, and H2O2 has a limited shelf life (probably something around 6 months). If your reactions stop working, try getting new H2O2).
Also note that ThermoFisher said that a solution of 50mM Tris pH 7.4 is a good amplification buffer.
a.The time you stain with the TSA reagent can be varied. Jie tried a lot of different times and found that 15 minutes usually worked well for Drosophila embryos. I found that for my biotin probes staining for 5-6 minutes worked better (gave a better signal to noise ratio).
23. Remove, rinse 1X with PBST and then wash 2X5 min with PBST.
Mounting
There are two options - Mount in 70% glycerol, 0.1M Tris pH 8.0 or mount in VectaShield. Vectashield contains anti-fade compounds so the fluorescence will last longer (up to a month).
a.Put two small coverslips on the ends of the slide. They will prevent the main coverslip from crushing the embryos. Cut the end off a blue tip to suck up the embryos. Place them in the middle of the slide. Remove as much PBST as possible with the 200ul pipet (careful to keep the tip presses lightly to the glass to prevent the embryos from getting sucked up) then place about 50 ul of glycerol solution near the embryos and cover with a large coverslip that sits on the small coverslips on the ends of the slide. To keep for more than a few days seal the sides of the large coverslip with nail polish. Store at 4oC.
b.If mounting in VectaShield, place the embryos on the slide (with small coverslips as above), then remove as much PBST as possible. Place a drop of VectaShield near the embryos, then cover with the large coverslip and seal as above. Store at 4oC.
Single fluorescent in situ hybridization using TSA (Tyramide Signal Amplification) kit
See also the notes on the colorometric staining protocol.
Day 1 Hybridization (~ 3 hrs)
1. Remove MeOH, and rinse 2X with MeOH.
2. Remove MeOH, rock 2X in MeOH, 5 min each time.
3. Remove, and rock 1X in PBST/MeOH 1:1 for 5 min.
4. Remove, and add 4% PFA, Rock for 25 min.
Turn on the heat block and set to 95oC.
Make hybridization buffer (HB). You need 2.5 ml/sample for 500ul rinses and washes.
5. Remove, rock in PBST 4X5 min.
6. Remove most of the PBST, heat the tube at 95℃ for 5 min.
7. Remove, and rock in pre-warmed HB/PBST 1:1 for 10 min at 60oC.
8. Remove, and add pre-warmed HB (works OK with RT HB), and incubate at 60oC for 30 min. Periodically invert the tube to break up clumps of embryos (not actually necessary usually).
9. Repeat Step 8.
10. Heat the probes (~ 0.2 ul of the stock in 50 ul of HB. or 1 ul for a new probe) at 90-95℃ for 5 min. Transfer it to ice immediately.
11. Remove HB and add the probe (50 ul). Flick gently the tube a few times and incubate at 60℃ overnight.
a.Probe concentrations must be determined empirically. I have had different probes work at concentrations from 0.1 ng/ul to 25 ng/ul. Jie said that all of her probes worked at the same concentration in TSA as in colorometric, but this was not true for mine, and there seemed to be no consistent pattern as to whether you would need more or less concentrated probe for TSA.
b.Biotin is probably not your best choice for a label as there is a lot of endogenous biotin around that gives a high background. You can mitigate this somewhat by staining for a shorter time (see below), but it’s probably better to use dig, fluorescein or DNP.
c.Leave the HB for tomorrow on the bench.
Day 2 Post-hybridization wash and primary antibody labeling (~ 2 hrs)
12. Remove probe without losing any embryo, and add 500 ul HB, rinse 1X5 min at 60oC degrees.
13. Remove, and add HB, rock for 30 min at 60 degrees.
14. Repeat Step 13.
15. Remove, and add pre-warmed HB/PBST 1:1, Rock for 10 min at 60 degrees.
16. Remove, and rinse 1X in PBST, wash 3X5 min in PBST at 60 degrees.
17. Remove, and add 250 ul of antibody diluted in PBST (1:250 anti-fluorescein, rabbit IgG or 1:250 anti-dig, mouse IgG). Incubate O/N at 4 degrees.
Day 3 HRP-conjugated secondary antibody and tyramide labeling , fluorescence detection (~ 5 hrs)
18. Remove primary antibody, and rinse once with PBST.
19. Remove, wash with PBST 4X15 min.
20. Remove, and add 1:100 HRP conjugated secondary antibody (100ul/sample), RT 2 hrs.
21. Remove, rinse 1X with PBST and then wash 4x15 min with PBST.
22. Remove, add 1:100 tyramide in amplification buffer working solution (add 1 ul of 30% H2O2 from the kit into 200 ul of amplification buffer from the kit to get 0.15% H2O2, then add 1:100 this intermediate dilution to amplification buffer to make 0.0015% H2O2), rock at RT for 15 min. (Wrap the tube with foil to block light).
Note: the amplification buffer working solution has to be made fresh before each use, and H2O2 has a limited shelf life (probably something around 6 months). If your reactions stop working, try getting new H2O2).
Also note that ThermoFisher said that a solution of 50mM Tris pH 7.4 is a good amplification buffer.
a.The time you stain with the TSA reagent can be varied. Jie tried a lot of different times and found that 15 minutes usually worked well for Drosophila embryos. I found that for my biotin probes staining for 5-6 minutes worked better (gave a better signal to noise ratio).
23. Remove, rinse 1X with PBST and then wash 2X5 min with PBST.
Mounting
There are two options - Mount in 70% glycerol, 0.1M Tris pH 8.0 or mount in VectaShield. Vectashield contains anti-fade compounds so the fluorescence will last longer (up to a month).
a.Put two small coverslips on the ends of the slide. They will prevent the main coverslip from crushing the embryos. Cut the end off a blue tip to suck up the embryos. Place them in the middle of the slide. Remove as much PBST as possible with the 200ul pipet (careful to keep the tip presses lightly to the glass to prevent the embryos from getting sucked up) then place about 50 ul of glycerol solution near the embryos and cover with a large coverslip that sits on the small coverslips on the ends of the slide. To keep for more than a few days seal the sides of the large coverslip with nail polish. Store at 4oC.
b.If mounting in VectaShield, place the embryos on the slide (with small coverslips as above), then remove as much PBST as possible. Place a drop of VectaShield near the embryos, then cover with the large coverslip and seal as above. Store at 4oC.