The protocol enables the completion of 7-color TSA multiplex IHC (DAPI + 6 biotinylated antibodies) within a single day. The click labeling of the antibodies is highly efficient and can be carried out at any volume, concentration, or buffer.
The kit includes StreptaClick®-HRP labeling reagent, the novel HRP block buffer for cyclic stainings on FFPE and frozen sections. The kit also includes a biotin block buffer used to block any remaining free StreptaClick® and Hydrogen peroxide.
Step-by-Step Multiplex Staining Protocol
Read the comprehensive user manual for detailed instructions.
Should you biotinylate the antibody ‘in house’ using a biotinylation kit, excess free biotin must be removed, potentially via a spin column, before the labeling process.
- Mix each biotinylated antibodies and StreptaClick®-HRP labeling reagent in separate tubes.
- Incubate for 10 minutes.
- Add biotin block buffer to each tube.
- Prepare the slide for immunostaining according to standard protocol. Avidin/biotin block is not needed.
- Block endogenous peroxide by adding a mixture of the provided HRP block buffer and Hydrogen Peroxide.
- Apply the first HRP-labeled antibody to your tissue sections and incubate 20-60 minutes at RT. Wash x3 in PBS.
- Dilute tyramide fluorochrome in tyramide amplification buffer and apply to your samples. Tyramide reagents are not provided in the kit but are also available for purchase in our online store. Incubate 10 minutes at RT and wash x2 in water or PBS.
- Apply the HRP block buffer/ hydrogen peroxide mixture and incubate for 12 minutes at RT.
- Repat step 6-8 for all HRP labeled antibodies.
- Wash, mount, and analyze under a fluorescence microscope.
Storage and Handling
Shipped in room temperature
Store at 4°C
StreptaClick® – HRP Workflow
Attach
Prep
Label each biotinylated antibody with StreptaClick®-HRP
TSA cycle
Step 1
Apply the first antibody to the tissue section (frozen or FFPE)
Step 2
Develop color with TSA reagents
Step 3
Quench the HRP enzyme with the novel HRP block buffer
