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
Back to Janos Luka's Home Page
Revised: June 5, 2000