This page contains some of the protocols we are using for immunostaining with our antibodies. In the near future it will also contain protocols used in molecular pathology.
- Antigen retrieval for immunostaining
- Rapid microwave enhanced immunostaining
- Silica Based DNA Purification from Various Sources
- Linear acrylamide as carrier for DNA precipitation
- DNA hybridization and detection by chemiluminescence
- DNA labeling using psoralen-biotin
- Method for direct cloning a PCR product, by the T-vector technique.
- Monoclonal antibody production (hybridoma development)
- DNA microarray Project and protocol (Restricted access/PASSWORD needed))
Rapid Immunofluorescence Staining using microwave oven
Microwave irradiation can accelerate antigen-antibody complex formation several fold. However, it is important that the temperature never reaches the level where the proteins, especially the antibodies, will denature. The protocol below was optimized for a 800 watts microwave oven with food rotator to distribute the microwave energy evenly. The water in the tray will adsorb the extra energy so the slides will not be heated over 37 C. The total staining time is approximately 15 minutes. The method is for fluorescence staining, but can be used for other type of staining by replacing the fluorescence secondary antibody with enzyme labelled antibody.
Equipment: GE countertop microwave oven Model JE425K (Dual Wave III oven) or equivalent, with rotator. Power: 800 Watts
- Fill a plastic tray (9" x 9"x 4") with 2 liter of cold tap water.
- Place the tray inside the microwave oven (center it).
- Add 20-30 microliter of diluted antibodies to each well on the slide.
- Place the slides on a rack and move it to the tray in the microwave oven (slides should be approximately 1/2 to 1 inch above the water.
- Set time on the microwave oven to 3 minutes and the power level to 7.
- Start the microwave oven and the rotator.
- When finished, let stand for 2 minutes in the microwave oven.
- Wash the slides in PBS for 2 minutes.
- Add 20-35 microliter of diluted FITC labelled secondary antibody to each well.
- Replace water in the tray with 2 liter of cold water.
- Place the slides on the rack and move it to the tray in the microwave oven.
- Set time on the microwave oven to 3 minutes and power to 7.
- Start the microwave heating and the rotator.
- When finished, let stand for 2 minutes in the microwave oven, then wash the slides in PBS for 3 minutes and 1 minute in Evan's blue. Mount the slides.
Antigen Retrieval for immunohistochemistry
The antigen retrieval technique was developed by Shi et al. in 1991. Antigen retrieval is a heat based antigen unmasking technique that can be used prior to immunohistochemical staining of archival formalin-fixed paraffin-embedded tissue sections. While some antibodies recognize the formalin-fixed antigen, the majority of monoclonal antibodies will not stain formalin-fixed tissues.
The method described here will unmask viral antigens in formaldehyde fixed tissues and they will be recognized by our monoclonal antibodies. Two methods will be presented. One based on microwave treatment, the other on autoclave heating.
Microwave method:
- Deparaffinize the section and rehydrate in PBS
- Immerse the slides in plastic tray containing 100 mM Tris-HCL pH 10.
- Adjust the power level on the microwave that the solution is just boiling.
- Microwave for 5 minutes at the predetermined power level.
- Check the level of buffer in the tray, fill up with buffer if necessary to cover the slides with buffer.
- Repeat step 4 and 5 three more times.
- Let the tray cool down at room temperature (takes 15-20 minutes, do not cool rapidly this may reduce the retrieval effect)
- Wash the slides twice in PBS for 2 minutes.
- The slides are ready for staining with standard procedure or with the microwave method
Autoclave method:
- Deparaffinize the section and rehydrate in PBS
- Immerse the slides in a autoclavable tray, containing 100 mM Tris-HCL pH 10.
- Autoclave at 120 C for 10 minutes.
- Let the tray cool down at room temperature.
- Wash the slides twice in PBS for 2 minutes
The slides are ready for staining with standard procedure or with the microwave method
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Revised: June 5, 2000