Private workspaceImmunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA) V.3

DOI
dx.doi.org/10.17504/protocols.io.95rh856
  • 1BioLegend
  • BioLegend
    Tech. support email: tech@biolegend.com
Sam Li
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DOI: dx.doi.org/10.17504/protocols.io.95rh856
External link: https://www.biolegend.com/protocols/immunohistochemistry-protocol-for-ultra-streptavidin-detection-kits-usa/4285/
Protocol Citation: Sam Li . Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io https://dx.doi.org/10.17504/protocols.io.95rh856
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: December 05, 2019
Last Modified: December 05, 2019
Protocol Integer ID: 30609
Keywords: IHC
Guidelines
Use with all Ultra Streptavidin Detection Kits.

All steps should be done in a humidity chamber such as 926301.

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.
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

  1. Xylene - 5 minutes in each of (3) different 250 mL containers
  2. 100% alcohol - 5 minutes in each of (3) different 250 mL containers
  3. 95% alcohol - 3 minutes in (1) 250 mL container
  4. 70% alcohol - 3 minutes in (1) 250 mL container
  5. Water -1 minutes in each of (3) different 250 mL containers
  6. #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)

  1. Heat slides in microwave on high for 1 minute 40 seconds in the appropriate retrieval solution at 1X.
  2. Reduce to low power and simmer 10 minutes in the microwave.
  3. Allow to cool on the bench top for 10 minutes.
  4. 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.

  1. 6 mL predilute antibodies are ready to use, do not dilute.
  2. 1 mL concentrate products can be diluted > 1:40 in PBS or other antibody diluent.
  3. 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.

  1. AEC Chromogen: 20 µL AEC chromogen + 1 mL AEC substrate buffer (1:50 Dilution)
  2. 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

  1. Submerge slides in Hematoxylin for 30 seconds (not provided).
  2. Rinse under running lab grade water for 1 minute or until water is clear.
  3. Submerge slides in Bluing Reagent for 1 minute (not provided).
  4. Rinse under running lab grade water for 1 minute.
Clear slides: Dehydrate the tissue.

  1. 95% alcohol 3 minutes in (1) 250 mL container
  2. 100% alcohol 5 minutes in each of (3) different 250 mL container
  3. 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.