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PELCO BioWave® Pro Microwave Tissue Processor

go to Confocal Microscopy and In Situ Hybridization Application Kit

In Situ Hybridization Protocols

In Situ Hybridization of mRNA and
Immunofluorescence using Microwave-assisted Processing

Protocol from Mark Sanders, Program Director, University Imaging Centers, University of Minnesota

Materials:

Procedure:
Sample preparation for Cultured Cells
1. Fix by adding 700 µl 37% formaldehyde. Incubate 6 minutes, 2 on-2 off-2 on, at 250 W on cold spot at room temp.
2. Wash 2x with 5 ml of 0.1M phosphate buffer and once in 5 ml solution P.
3. Resuspend in 1 ml solution P. Add b-mercaptoethanol and incubate at room temp for 10 min
4. Add 30 µl zymolyase. Incubate 8 minutes at 250 W on cold spot at room temp. Check cells under scope after 15 minutes. Cells lacking cell wall will be dark under phase scope.
5. While cells are being treated with zymolase, coat slide wells with 0.3% polylysine. Incubate 5 minutes at room temperature. Rinse 1x with water and air dry.
6. Resuspend in same volume of solution P
7. Apply 15-20 µl cell suspension to each well. Let settle to attach 15 minutes at room temperature
8. Gently aspirate off excess cells with filter paper.
9. Permeabilize cells with 0.5% NP-40 in solution P. Treat for 5 minutes.
10. While permeabilizing, make 0.1 M triethanolamine pH 8.0.
11. Aspirate off NP-40 and rinse 1x with solution P.
12. Equilibrate cells in freshly prepared 0.1 M triethanolamine pH 8.0 for 5 minutes at room temperature.
13. Block polar groups with 0.25% acetic anhydride/0.1 M TEA for 10 minutes at room temperature.
Hybridization: Beginning with this step, never let the wells dry out
14. Boil salmon sperm DNA for 5 minutes and put on ice for 5 minutes. Add salmon sperm DNA to 500 µg/ml in prehyb buffer (make just enough for prehybing).
15. Add 20 µl prehyb to each well. Prehyb 10 minutes at 400 W and 10 minutes at 0 power (off) at 37oC in humidified chamber on Coldspot.
16. Just before adding probe, boil salmon sperm DNA for 5 minutes and put on ice for 5 minutes. Add to 500 µg/ml in prehyb. Add digoxigenin-labeled probe to prehyb with salmon sperm DNA. Mix well.
17. Aspirate off prehyb and add 20 µl hybridization solution containing digoxigenin-labeled dT50 probe to each well. Hybridize for a total of 1 hour in cycles of 10 minutes 250W on/off in humidified chamber at 37°C.
18. Remove hybridization solution and wash wells:
 
  • 1x quickly with 2x SSC
  • 10 minutes at 250 W in 2x SSC at room temperature
  • 10 minutes at 250 W in 1x SSC at room temperature
  • 6 minutes at 250 W in 0.5x SSC at 37°C
  • 6 minutes at 250 W in 0.5x SSC at room temperature
19. Block cells in antibody blocking buffer for 6 minutes, 2'on-2'off-2'on, at 250 W at room temperature.
20. Add anti-digox Fab-FITC at 1:200 in antibody blocking buffer. 6 minutes, 2'on-2'off-2'on, at 250 W. If doing a double label with another antibody, dilute antibody appropriately and add here.
21. (If doing a double label) Wash 3x for 5 minutes each in antibody blocking buffer.
22. (If doing a double label) Add secondary antibody at desired dilution and incubate 6 minutes, 2'on-2'off-2'on, at 250 W at room temperature.
23. Wash wells with:
 
  • 1x quickly with Aby wash 1
  • Aby wash 1 for 10 minutes at room temperature
  • Aby wash 1 for 5 minutes, 2'on-1'off-2'on, at 250 W minutes at room temperature.
  • Aby wash 2 for 10 minutes at room temperature
  • Aby wash 2 for 5 minutes, 2'on-1'off-2'on, at 250 W minutes at room temperature.
Staining and Mounting Slides
24. Stain nuclei with DAPI solution for 5 minutes at room temperature.
25. Wash cells 2x with Aby wash 2 for 5 minutes each.
26. Air-dry slide and mount in mounting medium. Seal with clear nail polish.
Notes:
The probe is critical. The oligo should be of uniform length (ordered from DNA Express, 5' and 3' hydroxyl, RP-1 purified). The probe is digoxigenin-labeled using the Boehringer-Mannheim Genius-6ª kit. Each batch of probe should be titrated for optimal signal/noise ratio.

Immunofluorescence Protocol for BrdU

This microwave protocol is designed around the PELCO BioWave® Pro and the accessories included with the Immunolabeling Kit (36500-20). The specimen container is usually a 12 (Prod. #36170-12) or 24-well wellplate (Prod. #36172-24). Wellplate inserts (36170 and 36172) can be used for easy sample transfer between staining steps.

The rinse buffer can be 0.1M PBS or 0.1M TBS. This protocol was worked out with free-floating sections of rat brain approximately 60µm thick.
1. Buffer rinse : 3 x 40 sec. at 250W - no temperature control required.
2. Incubate tissue sections with 50% Formamide in 2x SSC: Depending on the wellplate or other container being used pick a microwave wattage setting (300-600W) that will produce final sample heating to 65ºC at the end of 5 minutes of microwave exposure. Periodically check the solution temperature over the 5 min. time span. It is important that reagent amounts are equal in all wells.
3. Rinse in 2x SSC: 2 x 40 sec. in the microwave at 150W.
4. Incubate in 2N HCl at 37ºC for 30 minutes in a shaker waterbath. No microwave parameters have been worked out for this step as yet.
5. Rinse in borate buffer: 1 x 40 sec. in the microwave at 150W.
6. Rinse in PBS or TBS: 5 x 40 sec. in the microwave at 150W
7. Blocking step: Use appropriate blocking buffer (PBS or TBS) containing from 2 to 10% normal serum and 0.25 to 1% Triton X-100: 2 min. on - 2 min. off - 2 min. on in the microwave at 200W.
8. Primary Antibody Incubation (Anti-BrdU): 3 min. on - 2 min. off - 3 min. on in the microwave at 150W. This step can be repeated if necessary depending on antibody dilution, source and other factors.
9. Buffer rinse: 2 x 1 min. in the microwave at 150W.
10. Secondary Antibody Incubation: 2 min. on - 2 min. off - 2 min. on in the microwave at 150W.
11. Buffer rinse: 2 x 1 min. in the microwave at 150W.
12. Float sections in 0.1M PO4 prior to mounting on slide.
13. Coverslip with appropriate mounting media and examine.

 


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Page Last Updated: May 19, 2011