Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits USA
Introduction
Use with all Ultra Streptavidin Detection Kits.
All steps should be done in a humidity chamber such as 926301.
Protocol Steps
- Clear Slides: Remove paraffin and hydrate the tissue.
Note: If using frozen sections, allow slides to come to room temperature for 15 minutes & proceed to step (F) only
- Xylene - 5 minutes in each of (3) different 250 mL containers
- 100% alcohol - 5 minutes in each of (3) different 250 mL containers
- 95% alcohol - 3 minutes in (1) 250 mL container
- 70% alcohol - 3 minutes in (1) 250 mL container
- Water -1 minutes in each of (3) different 250 mL containers
- #1 Blocking Reagent - 15 minutes using # 1 Blocking Reagent (clear in color) included in 50 test kit only, or 927401/927402 (sold separately) for 150 and 500 test kits.
- Rinse slides with lab grade water.
Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI). - Antigen Retrieval - Retrieve-ALL (927901, 928201, 927601) or Sodium Citrate (928502)
- Heat slides in microwave on high for 1 minute 40 seconds in the appropriate retrieval solution at 1X.
- Reduce to low power and simmer 10 minutes in the microwave.
- Allow to cool on the bench top for 10 minutes.
- Rinse with lab grade water.
- Antigen Retrieval - Rinse Slides with 1X PBS (926201)
Note: Other antigen retrievals could include EDTA, Proteinase K, Pepsin, protease VIII - follow antibody manufacturer's instructions. - Apply #2 Blocking Reagent (blue in color) for at least 5 minutes at room temperature. Do NOT wash after this step only.
- Blot off serum block.
- Apply primary antibody.
- 6 mL predilute antibodies are ready to use, do not dilute.
- 1 mL concentrate products can be diluted > 1:40 in PBS or other antibody diluent.
- If using a non-BioLegend antibody, dilute according to the manufacturer's instructions.
- Incubate primary antibody 20-60 minutes at room temperature (refer to incubation time listed on the datasheet).
- Rinse slides with 1X PBS.
- Apply #4 Linking Reagent (yellow in color) - and incubate slides for 20 minutes at room temperature.
- Rinse slides with 1X PBS.
- Apply #5 Labeling Reagent (orange in color) - and incubate slides for 20 minutes at room temperature.
- Rinse with 1X PBS.
- Apply chromogen and incubate slides for 5 minutes at room temperature.
- AEC Chromogen: 20 µL AEC chromogen + 1 mL AEC substrate buffer (1:50 Dilution)
- DAB Chromogen: 40 µL DAB chromogen + 1 mL DAB substrate buffer (1:25 Dilution)
Note: Not all USA Kits contain chromogen. If using a non-BioLegend chromogen, dilute and incubate according to the manufacturer’s instructions. To purchase separately from BioLegend, items are: AEC Chromogen 925804, AEC substrate buffer 925903; DAB Chromogen 926506, DAB Substrate Buffer 926605. If using a non-BioLegend chromogen, dilute and incubate according to the manufacturer’s instructions.
- Rinse slides with lab grade water.
- Counterstain
- Submerge slides in Hematoxylin for 30 seconds (not provided).
- Rinse under running lab grade water for 1 minute or until water is clear.
- Submerge slides in Bluing Reagent for 1 minute (not provided).
- Rinse under running lab grade water for 1 minute.
- Clear slides: Dehydrate the tissue.
- 95% alcohol 3 minutes in (1) 250 mL container
- 100% alcohol 5 minutes in each of (3) different 250 mL container
- Xylene 5 minutes in each of (3) different 250 mL container
- Coverslip
- Cover slip slide using Permanent Aqueous Mounting Medium or Xylene Based medium.
Note: Do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.
- Cover slip slide using Permanent Aqueous Mounting Medium or Xylene Based medium.
General Tips & FAQ
Tips:
- The multi-species system is suitable for use with primary antibodies that were raised in either mice or rabbits.
- The murine system is suitable for use with primary antibodies that were raised in mice.
- The rabbit system is suitable for use with primary antibodies that were raised in rabbits.
- Chromogen may be purchased separately.
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