Antigen Retrieval for immunohistochemistry -- ( Page 1, 2 )
Background: Formalin is a widely used fixative in pathology,
but it can form protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining
for immunohistochemical detection of certain antigens.
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 methods described here will unmask the antigens in formaldehyde fixed tissues. Two kind
of methods will be presented. One based on heating treatment, and the other on chemical treatment.
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
Sodium Citrate Antigen Retrieval:
- Place slides
in a glass slide holder and fill in the rest of the rack with blank
slides
(10 total) to ensure even heating.
- Place rack in 600 ml of 10 mM Sodium Citrate (pH 6.0, 100 mM stock) in a
glass 2L beaker. Mark a line at the top of the liquid on the beaker.
- Microwave for 20 minutes total, replacing evaporated water every
10 min.
- Cool slides for 20 minutes in the beaker.
- Wash 3 x 5¡ä in ddH2O, 1 x 5¡ä in 1X PBS.
Proteinase K Antigen Retrieval:
1.
Make a fresh solution of: 25 ul of 20 mg/ml Proteinase K
2.5 ml of 1 M Tris-Cl, pH 8.0
0.5 ml
of 0.5 M EDTA, pH 8.0
to 50 mls with ddH2O
2. Incubate slides in solution at 37¡ãC for 5 min (do NOT pre-warm Prot
K
solution). A Coplin staining jar works well for this step.
3. Wash 3 x 5¡ä with 1X PBS.
Urea Antigen Retrieval:
- Make
a fresh solution of 1 M urea
- Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 total) to
ensure even heating.
- Place rack in 600 ml of 1 M urea in a glass 2L beaker. Mark a line at the top of the liquid on the beaker.
- Microwave
for 10, 20 or 30 minutes total, replacing evaporated water every 5-10 minutes.
- Cool slides for 30 minutes to 1 hour in the beaker.
- Wash 3
x 5¡ä in ddH2O, 1 x 5¡ä in 1X PBS.
Trypsin antigen retrieval protocol
- Place slides or specimens into rack or other suitable
container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
- Incubate the specimens for 10 minutes at room temp
in PBS with occasional agitation to thoroughly hydrate the specimen.
- Pour off the PBS and incubate the specimen in a solution of 0.1%
trypsin (tissue culture grade), 0.1% calcium chloride, 20 mM Tris (pH 7.6-8.0) solution for 2-20 minutes at room temperature depending
upon the thickness and type of tissue. Optimization will be necessary. Typical time is 5 minutes.
- Stop the digestion by gently rinsing
the specimen under the cold tap for 5 minutes, followed by incubation in TBS or PBS.
- Samples are now ready for the addition of antibody.