In situ hybridization

The labeled nucleic acid single strand containing a specific sequence, that is, the probe, hybridizes under appropriate conditions with the complementary nucleic acid single strand, that is, the target nucleic acid, in cells, preserved tissue sections, or whole tissues, and then uses autoradiography or immunocytochemistry. The method detects labeled probes to display specific DNA or RNA molecules in situ in cells.

MCE has not independently verified the accuracy of these methods. They are for reference only.

Pick the slides and dry at room temperature for at least 30 minutes, secure the slides with 4% PFA on a shaker/shaker (best results: overnight at 4°C, 1 hour at room temperature is also OK)

1. Pre-hybridization and hybridization

Prepare 2.3% (meta)sodium iodate, prehybridization solution (prepared and preheated to 60°C), 0.1 M Tris pH 7.5 (prepare wash and borohydride - do not add borohydride yet), Proteinase K buffer Liquid preheated to 37°C, 20% acetic acid.

1) Rinse briefly 3 times in autoclaved water (quick rinse, few seconds, no timer). Keep 4% PFA refrigerated for step 11.

2) Incubate in 2.3% sodium iodate solution for 5 minutes at room temperature on a shaker.

3) Rinse briefly 3 times in autoclaved water.

4) Incubate in 20% acetic acid for 5 minutes at room temperature on a shaker.

5) Rinse briefly 3 times in autoclaved water.

6) Rinse 1 time in 0.1 M Tris pH 7.5.

7) Prepare sodium borohydride solution, incubate in 1% sodium borohydride (dissolved in 0.1M Tris pH 7.5) for 5 minutes.

8) Rinse briefly 4 times in autoclaved water. Prepare proteinase K solution by diluting 10 µL of proteinase K stock solution (10 mg/mL, stored at -20 °C) in 50 mL of proteinase K buffer (prewarmed at 37 °C). Shake well.

9) Incubate in Proteinase K for 5 minutes at 37°C.

10) Rinse 3 times with PBS (preferred) or autoclaved water.

11) Incubate in 4% PFA (saved from initial fixation step) for 5 minutes at room temperature.

12) Rinse briefly 3 times in autoclaved water.

13) Incubate in prehybridization solution for 2X 5 minutes at 60°C. Incubate once in solution, pour out, and incubate again. In this step, prepare the probe. Preheat the hybridization mixture to 60°C. Dilute digoxigenin-labeled RNA to 1ng/μL in the hybridization mixture. Prepare 100-150 μL per slide. Make sure the solution is mixed evenly.

14) Remove the slide from the jar and pat the edges with a paper towel to dry gently. Add approximately 100 μL of probe to the hybridization mixture for each slide. Hybridization mixture can be added directly to each section. Work quickly so the slices don't dry out completely.

15) Gently cover with glass coverslip. Place the coverslip carefully to avoid air bubbles.

16) Fill the slide bottom chamber with spent prehybridization mix (or 50% formamide in 1X saline solution).

17) Carefully place the slide into the chamber without disturbing the coverslip.

18) Seal the chamber in a plastic bag and incubate at 60°C overnight.

2. After hybridization

100 mL of 50% formamide in 1x salt solution (same as prehybridization solution). Warm to 60°C before use.

100 mL 0.5X saline solution (5 mL 10X saline solution + 95 mL autoclaved water). Warm to 60°C.

1) Carefully remove the coverslip and float it in the prehybridization solution, or lift it very gently from one corner. Transfer the slides back to the Wheaton jar.

2) Wash 2X in 50% formamide in 1x saline solution for 1 hour (2 hours total), store formamide waste at 60°C for next use.

3) Wash 2 times in 0.5X salt solution at 65°C for 1 hour each time (2 hours total) Use this time to prepare blocking solution. Store at 4°C until ready. Keeps for up to 1 week.

4) Wash 2X with 1X TBS pH 7.5 for 15 minutes at room temperature.

5) Block in blocking solution at room temperature for 1 hour. Fill the bottom of the slide chamber with dH₂O, tap the edge of the slide with a paper towel to dry it, outline the section with an immunohistochemistry pen, and add 40-50 μL of blocking solution per section

6) Dilute the anti-Dig-AP antibody in blocking solution to 1:1000 for overnight incubation (1:2000 for weekends and above), and prepare approximately 50 μL for each part.

7) Remove the blocking solution and add the antibody diluted in the blocking solution. Store at 4℃ in the chamber with water at the bottom.

3. Prepare NTMT solution (200 mL) Dissolve 25 mg of tetraimidazole hydrochloride in 50 mL of NTMT

1) Transfer slides back to Wheaton Jar taping off Antibody solution.

2) Wash 6X with 1X TBS pH 7.5 on a shaker at room temperature for 30 minutes.

3) Wash with tetraimidazole in NTMT for 15 minutes.

4) Wash 2 times for 15 minutes each in NTMT without tetraimidazole, dilute 500 μL NBT/BCIP solution in 49.5 mL NTMT before completing the second wash

5) Pour out the second washing liquid. Added NTMT and NBT/BCIP. Cover with parafilm and wrap Wheaton jars in foil to protect from light.

6) Monitor color response. Check at 10 minutes, 30 minutes, 1 hour, 2 hours, etc. until color develops. Some low-abundance probes take a long time to develop. These may need to be left at 4°C or room temperature overnight. This step varies greatly and requires the incorporation of actual probes.

7) Stop the reaction by washing 2X in 1X TBS for 5 minutes. If performing subsequent immunoassays, wash with 1X PBS instead and skip the following steps.

8) Dehydration through ethanol and xylene series. Water, water, 75% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, xylene (or substitute), xylene (or substitute), xylene (or substitute). at every step of the series. Dip the slide briefly 10 times.

9) Coverslip with Permount.

1. Always run the sense probe in parallel with the antisense probe to ensure specificity of the reaction.

2. All steps 1 and 2 should (ideally) be performed under RNase-free conditions.

3. Unlike many NBT/BCIP reagents, this color product is stable through ethanol/xylene dehydration and mounting procedures. However, it may fade after several months of storage and some crystalline precipitation may occur.