Multiplex IHC Detection kit (TSA amplification) is used for multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) on frozen tissue sections, which results in 10-50 times signal amplification compared with directly conjugated antibodies.
Images
Key facts
- Detection method
Fluorescent
- Assay type
Semi-quantitative
Reactivity data
| Application | Reactivity | Dilution info | Notes |
|---|---|---|---|
Application IHC-Fr | Reactivity Reacts | Dilution info - | Notes - |
Application IHC-P | Reactivity Reacts | Dilution info - | Notes - |
What's included?
Multiplex IHC Detection kit (TSA amplification) is used for multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) on frozen tissue sections, which results in 10-50 times signal amplification compared with directly conjugated antibodies.
Key facts
- Detection method
Fluorescent
- Assay type
Semi-quantitative
- Assay Platform
Microplate reader
Storage
- Shipped at conditions
Blue Ice
- Appropriate short-term storage conditions
+4°C
- Appropriate long-term storage conditions
+4°C
- Storage information
Please refer to protocols
Notes
Multiplex IHC Detection kit (TSA amplification) is used for multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) on frozen tissue sections, which results in 10-50 times signal amplification compared with directly conjugated antibodies. The kit attaches horseradish peroxidase (HRP) to biotinylated antibodies in solution with click reaction using a processed form of streptavidin conjugated with HRP. The HRP–labeled biotinylated antibodies can then be used for multiplex TSA immunostainings.
TSA reagents are not provided in the kit.
The kit contains a HRP block buffer optimized for preserving the morphology of frozen tissue sections. The HRP block buffer rapidly quenches the HRP enzymatic activity after each cycle. The fast staining protocol allows 7-color IHC (DAPI + 6 biotinylated antibodies) using TSA to be completed in one day. For 7-color IHC appropriate choice of tyramide fluorochromes as well as microscopy systems optimized for multicolor analyses are needed.
Use this kit to:
Multiplex IHC tyramide signal amplification (TSA) on frozen tissue sections, this kit can also be used on paraffin embedded sections.
Technical notes
The large kit contains reagents to perform 200 immunostainings (100 μl staining volume). The small kit contains reagents to perform 70 immunostainings. There is no heat treatment between staining cycles, which allows multiplex TSA to be performed on frozen sections with preserved morphology. The kit shall be used with biotinylated antibodies The kit can be combined with other immunostaining protocols, such as primary or secondary antibodies conjugated with fluorochromes.
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6 product images
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of mouse spleen tissue immunostained with biotinylated antibody against F4/80 (clone BM8) and Multiplex IHC Detection kit (TSA amplification) (ab312827). Left panel: Section stained with anti-F4/80 at cycle 1. Right panel: Section stained with anti-F4/80 at cycle 5. Fluorochrome: CF594 tyramide.
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of frozen mouse spleen tissue incubated with biotinylated antibody against F4/80 (clone BM8) labeled with Multiplex IHC Detection kit (TSA amplification) (ab312827). Before color development, one section was treated 12 minutes with HRP block buffer (left panel), while the control section was not treated (right panel). Both sections were then incubated 10 minutes with CF555 tyramide to develop color.
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of mouse spleen tissue stained with biotin rat-anti mouse CD4 (clone GK1.5) antibody at 1/400 dilution + Multiplex IHC Detection kit (TSA amplification) (ab312827) (left panel, exposure time 50 ms, x10 objective) or directly conjugated antibody (right panel, exposure time 700 ms, x10 objective). Followed by secondary antibody Alexa Fluor 488 rat-anti mouse CD4 (clone GK1.5) at 1/100 dilution
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of mouse small intestine tissue mounted in OCT compound, frozen down in liquid nitrogen, cut in 10 um thick sections, and stored at -20C until staining. The frozen tissue sections were fixed with acetone (30 seconds in ice cold 50% acetone/50%water + 10 min in ice cold 100% acetone), air dried, and washed in PBS. The fixed sections were immunostained using the Multiplex IHC Detection kit (TSA amplification) (ab312827) in combination with the following antibodies: biotin rat-anti CD31 (clone MEC13.3), biotin mouse-anti human alpha actin (clone 1A4), biotin rat-anti CD326 (clone G8.8), biotin rat-anti mouse CD8a (clone 53-6.7). TSA colors were developed with Alexa Fluor 488 tyramide, Cy3 tyramide, Alexa Fluor 594 tyramide, and Cy5 tyramide. Images were taken with 10x objective with a standard microscope equipped with appropriate set of filters (SpectraSplit7) for optimal fluorochrome separation. Exposure times (30-100 milliseconds).
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of mouse spleen tissue incubated in 4% paraformaldehyde/10% sucrose for 30 min at +20C, followed by incubation in 30% sucrose at +4C overnight. The samples were then mounted in OCT compound, frozen down in liquid nitrogen, cut in 10 um thick sections, and stored at -20C until staining. The frozen tissue sections were fixed with acetone (30 seconds in ice cold 50% acetone/50%water + 10 min in ice cold 100% acetone), air dried, and washed in PBS. The fixed sections were immunostained using the Multiplex IHC Detection kit (TSA amplification) (ab312827) in combination with the following antibodies: biotin rat-anti mouse B220 (clone RA3-6B2), biotin rat-anti mouse CD169 (clone 3D6.111), biotin rat-anti mouse CD4 (clone GK1.5), biotin rat-anti mouse CD8a (clone 53-6.7). TSA colors were developed with Opal tyramide dyes (Opal 520, Opal 570, Opal 620) and Cy5 tyramide. Images were taken with 10x objective with a standard microscope equipped with appropriate set of filters (SpectraSplit7) for optimal fluorochrome separation. Exposure times (30-100 milliseconds).
Immunohistochemistry (Frozen sections) - Multiplex IHC Detection kit (TSA amplification) (ab312827)
Immunohistochemical analysis of mouse spleen tissue incubated in 4% paraformaldehyde/10% sucrose for 30 min at +20C, followed by incubation in 30% sucrose at +4C overnight. The samples were then mounted in OCT compound, frozen down in liquid nitrogen, cut in 10 ?m thick sections, and stored at -20?C until staining. The tissue sections were fixed with acetone (30 seconds in ice cold 50% acetone/50%water + 10 min in ice cold 100% acetone), air dried, and washed in PBS. The fixed sections were immunostained using the Multiplex IHC Detection kit (TSA amplification) (ab312827) in combination with the following antibodies: biotin rat-anti mouse F4/80 (clone 53-6.7), biotin rat-anti mouse CD4 (clone GK1.5), biotin rat-anti mouse B220 (clone RA3-6B2), biotin rat-anti mouse CD8a (clone 53-6.7), biotin hamster-anti mouse (clone N418), and biotin rat-anti mouse Gr-1 biotin (clone RB6-8C5). The following tyramide dyes were used: CF430 tyramide, Alexa Fluor 488 tyramide, Cy3 tyramide, Alexa Fluor 594 tyramide, Cy5 tyramide, Opal polaris 780 tyramide kit. Images were taken with 10x objective with a standard microscope equipped with appropriate set of filters (SpectraSplit 7) for optimal fluorochrome separation. Exposure times (30-100 milliseconds).
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