User Manual StreptaClick®–HRP Multiplex IHC kit
Size:
Large 500 μl (200-250 stainings, 100 μL/staining)
Small 170 μl (70-90 stainings, 100 μL/staining)
Kit content and Storage:
StreptaClick®–HRP (antibody labeling reagent): +4ᵒC
Biotin block buffer: +4ᵒC
HRP block buffer: +4ᵒC
3% H2O2: +4ᵒC
Not provided in the kit
Tyramide fluorochromes
Tyramide amplification buffer
This document contains protocols for (A) antibody click labeling with horseradish peroxidase (HRP), and (B) the use of the HRP-labeled antibodies for tyramide signal amplification (TSA) multiplex immunostaining.
Introduction
The StreptaClick®–HRP Multiplex IHC kit provides a powerful method for multiplex immunohistochemistry using tyramide signal amplification (TSA), including frozen tissue sections. The method is based on horseradish peroxidase (HRP) that with a click reaction is attached to biotinylated antibodies using a processed form of streptavidin conjugated with HRP (StreptaClick®–HRP). The HRP–labeled biotinylated antibodies can then be used for multiple cycles of immunostainings where each fluorochrome is sequentially developed by TSA (TSA reagents are not provided). The kit contains a HRP block buffer optimized for preserving the morphology of frozen tissue sections. The HRP block buffer rapidly quenches the HRP enzyme at room temperature after each TSA cycle, and allows sequential multiplex IHC of up to six antibodies in one day.
Applications
Single and multiplex immunofluorescence staining of tissue sections using TSA to develop fluorescent color(s).
Before you begin
If the antibody is biotinylated ‘in house’ using a biotinylation kit, excess free biotin must be removed before use (e.g. by a spin column).
Do not use dry milk in the immunostaining buffer. It may contain free biotin that quenches StreptaClick®–HRP during the antibody labeling step. If desired, dry milk can be added after the HRP labeling reaction (Protocol A).
Dilute the biotinylated antibody with your immunostaining buffer of choice (e.g. PBS with 0.5% bovine serum albumin) before mixing with StreptaClick®–HRP. Pre-diluting to a working concentration avoids HRP quenching by sodium azide that is often used as a preservative in antibody stock solutions.
The antibody labeling reaction is not affected by BSA or other stabilizing proteins that may be present in antibody preparations.
The ratio between biotinylated antibody and StreptaClick®–HRP is important for optimal HRP labeling (Table 1). Make sure that you know the approximate antibody stock concentration. To avoid pipetting errors, use intermediate dilution if using less than 1 μl of the antibody stock solution.
Table 1. Volume of StreptaClick®–HRP and Biotin Block buffer for each μL antibody (antibody stock solution)
| Antibody stock concentration |
Antibody (μl) |
StreptaClick®-HRP (μl) |
Biotin block buffer (μl) |
| 1 mg/mL |
1 μL |
20 μL |
20 μL |
| 0.5 mg/mL |
1 μL |
10 μL |
10 μL |
| 0.1 mg/mL |
1 μL |
2 μL |
2 μL |
The antibody labeling is performed at room temperature (RT). Multiple biotinylated antibodies can be labeled with HRP in parallel. Store the HRP-labeled antibodies at +4ᵒC, and use them for TSA immunostaining within 8 hours.
(A) HRP labeling protocol
The HRP labeling protocol attaches HRP to biotinylated antibodies that will be used for TSA immunostaining.
- Put two tubes of the same size in a rack (e.g. 1.5 ml microfuge tubes).
- In the first tube (Tube A), prepare your antibody working solution. As a guidance, use a 1:500-1:2000 dilution from a 1 mg/ml antibody stock solution. Avoid pipetting antibody volumes of <1 μl. Avoid dry milk in the immunostaining buffer, since it may contain free biotin.
- To the second tube (Tube B), add the appropriate amount of StreptaClick®–HRP according to Table 1.
- Transfer all antibody solution from Tube A to the second tube (Tube B) and mix immediately by pipetting up and down. Avoid bubbles. The transfer from Tube A to Tube B is important for and even distribution of HRP bound to the antibodies.
- After 10 minutes or more, add Biotin block buffer according to Table 1 and mix. The Biotin block buffer immediately inactivates any remains of active StreptClick–HRP. The biotinylated antibody is now labeled with HRP and ready to be used for TSA immunostaining.
(B) TSA immunostaining protocol
The HRP-labeled antibodies can be used for multiplex TSA immunostaining. The protocol does not use heat treatment between cycles, which allows TSA multiplex immunostaining also on frozen tissue sections. Each staining cycle contains three main procedures – Antibody incubation, Color development, and HRP block.
- Prepare your tissue sections for immunostaining according to standard protocols. There is no need for avidin/biotin blocking.
- Block endogenous peroxidases with the provided HRP block buffer. Add 1 μl 3% H2O2 to each 100 μl of HRP block buffer and apply to the tissue sections. Incubate for 12 minutes at RT. The HRP block buffer/H2O2 will also be used in step 5 and can be stored at +4ᵒC in the dark for 24 hours.
- Apply 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. Incubate 10 minutes at RT and wash x2 in water or PBS.
- Apply HRP block buffer/H2O2 to the tissue sections and incubate for 12 minutes at RT. This step is only needed when a sequential immunostaining cycle will be performed. Wash x3 in PBS.
- Repeat steps 3-5 with the next HRP-labeled antibody.
- Wash, mount, and analyze under a fluorescence microscope.
Trouble shooting guide
Weak/No signals
– Check that your biotinylated antibody works using a two-step immunostaining with the StreptaClick®–HRP; 1) Incubate tissue section with the biotinylated antibody. 2) Wash and incubate tissue sections with StreptaClick®–HRP alone diluted 1:20 in PBS. Wash and develop color with TSA.
– The ratio between biotinylated antibody and StreptaClick®–HRP is important. Check the concentration of the antibody. If you do not know the exact concentration, test different amounts of labeling reagent during antibody labeling.
– It is important to first prepare an antibody working solution before mixing with StreptaClick®–HRP. Pre-diluting to a working concentration avoids HRP quenching by sodium azide that is often used as a preservative in antibody stock solutions.
– Fast mix between the antibody working solution and the StreptaClick®-HRP is important. Do not add the StreptaClick®-HRP reagent into the tube of antibody working solution (Tube A). Make a full transfer of the antibody working solution (Tube A) into the tube of StreptaClick®-HRP reagent (Tube B).
– Increase the amount of antibody used during the immunostaining step.
– Increase the amount of tyramide fluorochrome during the color developing step. Twice the amount of tyramide fluorochrome can improve the signal significantly.
– The HRP block buffer may not have been washed away properly before adding the next HRP–labeled antibody to the sample.
– Some antibodies have biotin conjugated near the antigen-binding site, resulting in sterical hindrance when attaching the HRP to the antibody. Using less StreptaClick®–HRP during the labeling step may help.
– Do not use dry milk in the immunostaining buffer, since it may contain free biotin that quenches the StreptaClick®–HRP. If desired, dry milk can be added after the antibody labeling reaction.
Cross-over signal from other antibodies
– The biotin block step may be incomplete. Check that the Biotin block buffer is properly added before applying HRP-labeled antibodies to the tissue.
– The HRP block step may be incomplete. Check that H2O2 is properly added to the HRP block buffer. Prolong the incubation time to 15-18 min.
To strong signals or unspecific signals
– Lower the amount of antibody used during the immunostaining step.
– Decrease the amount of tyramide fluorochrome during the color developing step.
Store Tyramide Amplification Buffer Plus at +4°C. Protect from light. Product is stable for at least 6 months from date of receipt when stored as recommended. Warm 1X Tyramide Amplification Buffer 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.
Store the tyramide dyes at -20°C, protected from light. Product is stable for at least 12 months from date of receipt. Before use, dissolve each dye with 200 μl DMSO. Aliquot in vials and store at ≤ -20°C. The aliquots can be stored in the freezer for at least 12 months. Avoid freeze-thaw cycles. However, a vial can tolerate one freeze-thaw cycle.
Fluorescence tyramide dyes are used for tyramide signal amplification (TSA) to generate strong fluorescence signals in immunohistochemistry (IHC), immunocytochemistry (ICC) and in situ hybridization (FISH). TSA is based on the ability of horseradish peroxidase (HRP), in the presence of low concentrations of hydrogen peroxide, to convert tyramide-containing dyes into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP. This leads to significant amplification of the signal at the target.
Multiple TSA procedures can be performed sequentially to label different targets in the same sample, by performing HRP quenching or antibody stripping after each tyramide reaction. Since the tyramide dye is covalently attached to the sample it will remain.
The samples are now ready for fluorescence imaging or for another round of multistaining.
