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 50 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

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 30% H₂O₂ and Block Activator, it can be stored at room temperature for up to 24 hours.

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. 

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

1. 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 500 μL 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 the appropriate amount of StreptaClick®–HRP according to the ratios in Table 1. 
4. Transfer all the contents in tube A to tube B and mix immediately by pipetting up and down. Avoid bubbles. This step is important for an even distribution of HRP bound to the antibodies.
5. Incubate at room temperature for at least 12 minutes and add Biotin block buffer according to Table 1 and mix. The Biotin Block Buffer immediately inactivates any remaining active 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 8 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 8 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. Block endogenous peroxidases with Activated HRP Block Buffer. Prepare desired amount of Activated HRP Block Buffer by adding 10 μL of 3% H₂O₂ and 50 μL Block Activator to each 1 mL of HRP Block Buffer.

9. Apply the Activated HRP Block Buffer to the tissue sections and incubate for 12 minutes at RT. 
10. Gently wash the sample twice in distilled water and then once in 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. While the tissue incubates with the HRP-labeled antibody, determine the total volume of staining solution needed per corresponding Tyramide dye.
13. Working in a chemical fume hood, prepare 0.15% H₂O₂ by adding 5 µL of 30% H₂O₂ in 1000 µL of DI water.
14. Mix 5 µL of 0.15% H₂O₂ with 490 µL TSA Amplification Buffer Plus.
15. Add 5 µL of Tyramide Dye and mix well.
16. Following step 11, apply the prepared Tyramide fluorochrome to your samples. Incubate 10 minutes at room temperature.
17. Wash twice in distilled water.

18. Apply the activated HRP Block Buffer (from step 8) to the tissue sections and incubate for 12 minutes at RT. This step is only needed when a sequential immunostaining cycle will be performed.
19. Wash x2 in distilled water and x1 in PBS.
20. Repeat steps 5-11 with the next HRP-labeled antibody.
21. Wash twice in distilled water, mount, and analyze under a fluorescence microscope.