Multiplex Immunohistochemistry Kit (3-color TSA) Protocol

Introduction

This protocol is intended to be used with BioLegend’s Multiplex Immunohistochemistry Kit (3-color TSA) (Cat. No. 426503/426504). 

Kit sizing and capacity

This kit is conveniently supplied in 90 and 250 test sizes. A single test is defined as 100 µL of staining volume on frozen or formalin-fixed paraffin-embedded (FFPE) tissues.

Kit contents (90/250 Tests Size)

The following items should be stored at 4°C

• 3% H₂O₂ (300 µL) 
• Biotin Block Buffer (550 µL) 
• HRP Block Buffer (9/25 mL)
• Block Activator (1450 µL)
• StreptaClick^®–HRP (170/500 µL) 
• TSA Amplification Buffer Plus (25 mL) 
• 30% H₂O₂ (100 µL) 

• Tyramide 488 (80 µg)
• Tyramide 555 (80 µg) 
• Tyramide 647 (80 µg) ​

Materials Not Provided

• Distilled water
• PBS
• Desired antibodies (biotin labeled)
• Desired staining/immunostaining buffer
• DMSO

General Considerations

• If user is performing ‘in-house’ biotin labeling of the antibody using a biotinylation kit, we recommend removing any excess biotin that may be present in the antibody stock solution (e.g. by using a spin column).
• Do not use dry milk in the immunostaining buffer as it may contain free biotin. If desired, dry milk can be added after the HRP labeling reaction.
• Dilute the biotinylated antibody with your staining buffer of choice before mixing with StreptaClick^®–HRP. Pre-diluting your antibody avoids HRP quenching by sodium azide, which is often used as a preservative in antibody stock solutions.
• BSA or other stabilizers in antibody preparations do not inhibit the antibody labeling reaction.
• The ratio of antibody to StreptaClick^®–HRP is important for optimal HRP labeling (Table 1). User must know the approximate antibody stock concentration. To avoid pipetting errors, use an intermediate dilution if using less than 5 μL of the antibody stock solution.
• The antibody labeling is performed at room temperature allowing multiple biotinylated antibodies to be labeled with HRP in parallel. Store the HRP-labeled antibodies at 4°C and use them for TSA immunostaining within 8 hours.
• Once the activated HRP Block Buffer is prepared with 3% H₂O₂ and Block Activator, it can be stored at room temperature for up to 24 hours.
• The Tyramide Amplification Buffer Plus and 30% Hydrogen peroxide should be stored at +4°C protected from light. For use, warm Tyramide Amplification Buffer Plus to room temperature and mix well by vortexing or shaking to make sure all solids are completely dissolved before each use. The buffer can be warmed in a 37°C water bath for convenience.
• It is important to wash in water (and not PBS) before applying the HRP block buffer.

Prepare Tyramide Dye Stock Solution

Upon arrival, store the dyes at -20°C, protected from light. Before use, dissolve each vial in 100 µL DMSO + 100 µL PBS. Aliquot in vials and store at ≤ -20°C. The aliquots can be stored in the freezer for at least 12 months. Once thawed, store at +4°C and use within a week.

HRP Labeling Protocol

This protocol provides instructions to label HRP to the biotinylated antibody of choice. Once this HRP labeling procedure is complete, the HRP labeled antibody can then be used for TSA Immunostaining. 

1. For each antibody, obtain two tubes of the same size (will vary depending on desired volume for HRP labeling) and label as tube A and B. 
2. In tube A, add the desired volume (~100 µL per section depending on tissue) of your staining buffer of choice such as 5% FBS in PBS. Then, add the desired amount of biotinylated antibody to reach the working concentration for immunostaining. 

3. In Tube B, add 20 µL of StreptaClick®-HRP for every 1 µg of primary antibody in Tube A. Scale volume of StreptaClick®-HRP as needed based on antibody amount. See Table 1 for examples.

Table 1. Volume of StreptaClick^®–HRP and Biotin Block buffer per 1 μL antibody stock solution

4. Transfer all the contents in tube A to tube B and mix immediately by pipetting rapidly up and down while avoiding bubbles. This step is important for an even distribution of HRP bound to the antibodies. Incubate for at least 10 minutes at room temperature.
5. Add an equal volume of Biotin Block Buffer as you did for the StreptaClick®-HRP and mix. See Table 1 for reference. The Biotin Block Buffer immediately inactivates any unbound StreptaClick®-HRP.”
6. The biotinylated antibody is now labeled with HRP and ready to be used for TSA immunostaining. If not immediately proceeding to TSA immunostaining, store the HRP-labeled antibody at +4°C and proceed with TSA immunostaining within 12 hours.

TSA Immunostaining Protocol

This protocol provides instructions for single or multiplex TSA immunohistochemistry on frozen and FFPE tissue sections. Proceed to this protocol within 12 hours of completing the HRP Labeling Protocol. This kit does not require heat treatment between cycles, which allows for TSA immunostaining on frozen tissue sections in addition to FFPE sections. Each staining cycle contains three main procedures - Antibody incubation, color development, and HRP block.

7. Prepare your tissue sections for immunostaining according to standard protocols. There is no need for avidin/biotin blocking.
8. Determine the total amount of Activated HRP Block Buffer needed for your experiment. You will need apply ~50 µL of Activated HRP Block Buffer to each section between each staining cycle to reduce background signal from endogenous peroxidases (step 18).

   Example: For 3-color staining on 10 sections = 3 antibodies × 10 sections × 50 µL = 1.5 mL total of Activated HRP Block Buffer

   To prepare 1 mL Activated HRP Block Buffer, add and mix:

   • 1 mL of HRP Block Buffer
   • 10 µL of 3% H₂O₂
   • 50 µL Block Activator

9. Wash the tissue briefly with water if there is residual PBS before applying the Activated HRP Block Buffer. Apply the Activated HRP Block Buffer to the tissue sections and incubate for 15 minutes at room temperature.
10. Gently wash the sample 3 times with PBS.
11. Apply the first of your HRP-labeled antibody (from step 6 of HRP Labeling Protocol) to your tissue sections and incubate 20-60 minutes at RT. After incubation, wash 3 times in PBS.
12. You may prepare the Tyramide dye working solutions while the tissue incubates with the HRP-labeled antibody in the step above. Determine the total volume of Tyramide solution needed per corresponding Tyramide dye.
13. Tyramide dye color development.

    Prepare an intermediate 0.15% H₂O₂ solution:

    For every 1 mL of DI water, add 5 µL of 30% H₂O₂

     

    Then, prepare the desired volume of each Tyramide dye working solution:

     

    For every 1mL of Tyramide dye working solution desired, add:

     

    • 980 µL TSA Amplification Buffer Plus
    • 10 µL of 0.15% H₂O₂ solution
    • 10 µL of desired Tyramide Dye Stock Solution

    Once step 11 incubation and wash are completed, apply the Tyramide dye working solution to the tissue section. Ensure you are using the Tyramide dye which is desired for the antibody used in this cycle (user preference). Incubate for 10 minutes at room temperature protected from light.

     

    Tip: The color development time may require optimization (typically 10-20 min). The working concentration of the Tyramide dyes may be adjusted to achieve the desired signal intensity. The typical dilution range is 1:50-1:200.

     

14. Wash 3 times in distilled water.
15. Repeat steps 8-14 (HRP Block > wash > add HRP-labeled antibody > wash > tyramide color development > wash) with the next HRP-labeled antibody from step 6.
16. Wash twice in distilled water, mount, and analyze under a fluorescence microscope.