Figure 2. Illustration of the cyclic staining process using Aluora spatial amplification. The protocol begins with antigen retrieval followed by blocking of nonspecific epitopes. The tissue can then be labeled with the primary antibody followed by the poly-HRP secondary reagent. Finally, the addition of the Invitrogen Aluora dye will covalently deposit the fluorophore onto the tissue. This process can be repeated for each primary antibody starting from Step 1 where the antibody complex will be removed.
Invitrogen Aluora Spatial Amplification Kits offer a powerful method for detecting low-abundance targets in multiplex immunohistochemistry (mIHC) applications. A limitation to mIHC, spatial proteomics, and other multiplex imaging experiments is detecting low-abundance proteins over autofluorescence and non-specific background. Our menu of Aluora Spatial Amplification Kits help overcome this limitation with bright Aluora dyes and enzyme-mediated signal amplification.
What is Aluora spatial amplification?
By combining intense Aluora dyes with enzyme-mediated signal amplification, Aluora Spatial Amplification Kits produce exceptionally bright, covalently attached fluorophores for multiplex IHC and spatial imaging. Aluora Spatial Amplification Kits enhance fluorescence signal intensity to facilitate the visualization of up to eight targets in one tissue sample. This technique is invaluable for spatial biology research, allowing for the detection and understanding of spatial relationships and cellular interactions.
The basic steps for using Aluora spatial amplification involve primary antibody labeling, poly-horseradish peroxidase (HRP) secondary reagent detection, fluorophore deposition through the Aluora dye reaction and finally stripping of the antibody complex leaving the fluorophore covalently labeled to the tissue sample.
Figure 1. Aluora signal amplification labeling workflow. Tissue is labeled with a primary antibody for the target of interest and then incubated with a poly-horseradish peroxidase (HRP) secondary reagent. The Aluora dye reaction then covalently deposits the fluorophore onto the tissue. The antibody complex can then be stripped, and the protocol can be repeated for multiplex imaging.
Benefits of Aluora Spatial Amplification Kits and reagents
Invitrogen Aluora dyes offer enhanced brightness, enabling improved sensitivity making them excellent for spatial proteomics applications. With eight different fluorophores of similar brightness and distinct emission spectra, up to 8-plex multiplexing is achievable on spectral imaging systems, allowing for comprehensive spatial imaging. These fluorophores also enable exceptional detection of low-abundance targets, due to enzyme-mediated signal amplification, and require significantly less primary antibody. Primary antibodies from the same species can also be used with these reagents with the cyclic stripping of antibodies between rounds of signal amplification or with an automated slide stainer like the Leica BOND, enabling streamlined multiplex staining workflows.
- Enhanced brightness—experience improved fluorescence sensitivity with brighter fluorophores, optimized for spatial proteomics applications, outperforms other signal amplification systems to enable primary antibody dilutions of 1:1,000 to 1:50,000
- Increased detection of low abundance targets—achieve exceptional detection of extremely low-abundance targets over background noise due to the HRP enzyme-mediated Aluora signal amplification
- Seamless instrument compatibility—specifically designed for fluorescence spectral imaging on the EVOS S1000 Spatial Imaging System and Akoya PhenoImager, ensuring seamless integration with spatial imaging systems
- Expanded antibody options for multiplexing—enzyme-mediated labeling via covalent attachment of the fluorophore allows for primary antibody stripping in between rounds of signal amplification and enables the use of primary antibodies from the same host species, expanding the antibody options for multiplex labeling experiments
How to use Aluora Spatial Amplification Kits for spatial imaging
The Aluora spatial amplification reagents include a set of eight fluorescent dyes specifically designed for spatial imaging. These dyes enable up to 8-plex multiplexing and have emission spectra that can be spectrally unmixed for multiplexed spatial imaging on the EVOS S1000 Spatial Imaging System and other systems such as the Akoya PhenoImager. This simplifies panel design and assay development. Additionally, the Aluora dyes can be used on any imaging system equipped with standard filter sets although high-plex panels require careful consideration of spectral overlap.
Multiplexing with Aluora Spatial Amplification Kits
The ability to perform mIHC using multiple primary antibodies from the same host species opens new possibilities for multiplex labeling. Aluora spatial amplification reagents not only provide enhanced sensitivity of low-abundance targets, but also allow multiplex detection of primary antibodies from the same or different host species on a tissue sample without risking antibody cross-reactivity. The Aluora spatial amplification technology produces highly reactive tyramide-like radicals that covalently bind and localize to antibody staining in IHC samples. Primary and secondary antibodies can be removed from the tissue and using antibody stripping after the Aluora dye signal development has completed without decreasing the intensity of the fluorophore deposited. Once antibodies are removed, the tissue can be reprobed with a primary antibody from the same or different host species (raised in rabbit or mouse) without risk of cross-reactivity, followed by detection with another round of Aluora spatial amplification. For added convenience and higher throughput, these steps can be performed on an automated slide stainer such as the Leica Bond RX systems.
Aluora spatial amplification reagents selection guide
| Aluora 430 Spatial Reagent | Aluora 488 Spatial Reagent | Aluora 514 Spatial Reagent | Aluora 555 Spatial Reagent | Aluora 594 Spatial Reagent | Aluora 647 Spatial Reagent | Aluora 700 Spatial Reagent | Aluora 750 Spatial Reagent* | |
| Detection | Fluorescence signal amplification of 100X | |||||||
| Ex/Em | 427/499 | 493/518 | 512/529 | 553/567 | 589/615 | 652/670 | 687/706 | 757/783 |
| Channel | 430 | 488 | 514 | 555 | 594 | 647 | 700 | 750 |
| EVOS filter | CFP | GFP | YFP | RFP | Texas Red | Cy5 | Cy5.5 | Cy7 |
| Sensitivity | Excellent | |||||||
| Application | Multiplex staining up to 8-plex for fluorescence spectral and spatial imaging applications | |||||||
| Automation | Compatible with multiplex staining on automated slide stainers such as Leica Bond | |||||||
| Individual Aluora reagents* | AS430HRP | AS488HRP | AS514HRP | AS555HRP | AS594HRP | AS647HRP | AS700HRP | AS750HRP |
| Aluora Goat anti-Mouse HRP Kits** | A40001329 | A40001330 | A40001331 | A40001332 | A40001333 | A40001334 | A40001335 | A40001336 |
| Aluora Goat anti-rabbit HRP Kits** | A40001337 | A40001338 | A40001339 | A40001340 | A40001341 | A40001342 | A40001343 | A40001344 |
| Aluora Streptavidin HRP Kits** | A40001345 | A40001346 | A40001347 | A40001348 | A40001349 | A40001350 | A40001351 | A40001352 |
| Aluora Spatial Rainbow Kit | A40002450 | |||||||
* The Aluora 750 Spatial Reagents are near IR tyramide-like reagents that covalently attach to tissue in a single step. Enzyme inactivation and antibody stripping can be immediately performed for subsequent signal amplification reactions, offering an advantage over the other near-IR signal amplification reagents on the market for spatial biology. ** Aluora spatial amplification kits contain all the necessary components to perform the assay. Multiplex experiments can be performed by purchasing additional Aluora spatial reagents. | ||||||||
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Data image gallery from Aluora spatial amplification experiments
Figure 3. Invasive ductal carcinoma tissue stained with the Aluora Spatial Rainbow Kit and DAPI. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora Spatial Rainbow Kit (Cat. No. A40002450). Composite image with zoomed inset. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 4. Invasive ductal carcinoma tissue stained with the 8-plex Aluora spatial amplification assay and DAPI shown in individual tiles. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora Spatial Rainbow Kit (Cat. No. A40002450). Tiles represent individual Aluora dye colors. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 5. Single cell segmentation and phenotyping reveal spatial distribution of immune cell subpopulations. Human invasive ductal carcinoma of breast tissue analyzed to identify single cell target expression and specific localization of immune cell subpopulations. (A) Composite image using the Aluora Spatial Rainbow Kit (Cat. No. A40002450). (B) Tissue segmentation identifying vimentin positive and negative regions. (C) 0.7 mm2 area analyzed containing 10,454 cells, with white cell segments indicating dual positive proliferating & immune cell phenotypes. (D) Identification of region-specific cell localization. Analysis of the multiplex immunofluorescence stitched image was performed on the Indica Labs HALO (version 4.0.5107.318) software.
Figure 6. Invasive ductal carcinoma tissue stained with the 8-plex Aluora spatial amplification assay and DAPI. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora spatial amplification 8-plex assay. Composite image shown on the left and individual unmixed channels shown on the right. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 7. Aluora 594 Spatial Dye demonstrates excellent signal compared to Competitor O and secondary antibody equivalents with lower primary antibody concentrations. Human FFPE small intestine was incubated with 1:10,000 of PCNA Monoclonal Antibody (PC10) (Cat. No. 13-3900) then labeled with either Alexa Fluor 594 Secondary Antibody (Cat. No. A-11014), the reagents in the Aluora 594 Goat anti-Mouse HRP Kit (Cat. No. A40001331), or fluorophore equivalent of competitor 1. N=5 images analyzed for signal to background, P < 0.0001. Images were captured with an Invitrogen EVOS S1000 Spatial Imaging System.
Figure 8. Aluora spatial amplification reagents in human FFPE small intestine. Human FFPE small intestine was incubated with Mouse PCNA antibody (PC10, Cat. No. 13-3900) and labeled with Aluora 430 Goat anti-Mouse HRP Kit (Cat. No. A40001329) seen in green, Mouse Vimentin antibody (V9, Cat. No. MA5-11883) and labeled with Aluora 594 Goat anti-Mouse HRP Kit (Cat. No. A40001333) seen in red and Mouse SMA antibody (1A4, Cat. No. 14-9760-82) and labeled with Aluora 700 Goat anti-Mouse HRP Kit (Cat. No. A40001336) seen in white, and a DAPI nuclear stain. The slides were mounted in Prolong Glass Antifade Mountant. Images were captured with an Invitrogen EVOS M7000 Imaging System (see information about EVOS Imaging Systems).
Figure 9. Aluora spatial amplification reagents in human FFPE small intestine. Human FFPE small intestine was incubated with Mouse PCNA antibody (PC10, Cat. No. 13-3900) and labeled with Aluora 647 Goat anti-Mouse HRP Kit (Cat. No. A40001334) seen in red, Mouse Vimentin antibody (V9, Cat. No. MA5-11883) and detected with Aluora 488 Goat anti-Mouse HRP Kit (A40001330) seen in green and Mouse SMA antibody (1A4, Cat. No. 14-9760-82) and detected with Aluora 700 Goat anti-Mouse HRP Kit (Cat. No. A40001336) seen in white and a DAPI nuclear stain. The slides were mounted in Prolong Glass Antifade Mountant. Images were captured with an Invitrogen EVOS M7000 Imaging System (see information about EVOS Imaging Systems).
What is Aluora spatial amplification?
By combining intense Aluora dyes with enzyme-mediated signal amplification, Aluora Spatial Amplification Kits produce exceptionally bright, covalently attached fluorophores for multiplex IHC and spatial imaging. Aluora Spatial Amplification Kits enhance fluorescence signal intensity to facilitate the visualization of up to eight targets in one tissue sample. This technique is invaluable for spatial biology research, allowing for the detection and understanding of spatial relationships and cellular interactions.
The basic steps for using Aluora spatial amplification involve primary antibody labeling, poly-horseradish peroxidase (HRP) secondary reagent detection, fluorophore deposition through the Aluora dye reaction and finally stripping of the antibody complex leaving the fluorophore covalently labeled to the tissue sample.
Figure 1. Aluora signal amplification labeling workflow. Tissue is labeled with a primary antibody for the target of interest and then incubated with a poly-horseradish peroxidase (HRP) secondary reagent. The Aluora dye reaction then covalently deposits the fluorophore onto the tissue. The antibody complex can then be stripped, and the protocol can be repeated for multiplex imaging.
Benefits of Aluora Spatial Amplification Kits and reagents
Invitrogen Aluora dyes offer enhanced brightness, enabling improved sensitivity making them excellent for spatial proteomics applications. With eight different fluorophores of similar brightness and distinct emission spectra, up to 8-plex multiplexing is achievable on spectral imaging systems, allowing for comprehensive spatial imaging. These fluorophores also enable exceptional detection of low-abundance targets, due to enzyme-mediated signal amplification, and require significantly less primary antibody. Primary antibodies from the same species can also be used with these reagents with the cyclic stripping of antibodies between rounds of signal amplification or with an automated slide stainer like the Leica BOND, enabling streamlined multiplex staining workflows.
- Enhanced brightness—experience improved fluorescence sensitivity with brighter fluorophores, optimized for spatial proteomics applications, outperforms other signal amplification systems to enable primary antibody dilutions of 1:1,000 to 1:50,000
- Increased detection of low abundance targets—achieve exceptional detection of extremely low-abundance targets over background noise due to the HRP enzyme-mediated Aluora signal amplification
- Seamless instrument compatibility—specifically designed for fluorescence spectral imaging on the EVOS S1000 Spatial Imaging System and Akoya PhenoImager, ensuring seamless integration with spatial imaging systems
- Expanded antibody options for multiplexing—enzyme-mediated labeling via covalent attachment of the fluorophore allows for primary antibody stripping in between rounds of signal amplification and enables the use of primary antibodies from the same host species, expanding the antibody options for multiplex labeling experiments
How to use Aluora Spatial Amplification Kits for spatial imaging
The Aluora spatial amplification reagents include a set of eight fluorescent dyes specifically designed for spatial imaging. These dyes enable up to 8-plex multiplexing and have emission spectra that can be spectrally unmixed for multiplexed spatial imaging on the EVOS S1000 Spatial Imaging System and other systems such as the Akoya PhenoImager. This simplifies panel design and assay development. Additionally, the Aluora dyes can be used on any imaging system equipped with standard filter sets although high-plex panels require careful consideration of spectral overlap.
Multiplexing with Aluora Spatial Amplification Kits
The ability to perform mIHC using multiple primary antibodies from the same host species opens new possibilities for multiplex labeling. Aluora spatial amplification reagents not only provide enhanced sensitivity of low-abundance targets, but also allow multiplex detection of primary antibodies from the same or different host species on a tissue sample without risking antibody cross-reactivity. The Aluora spatial amplification technology produces highly reactive tyramide-like radicals that covalently bind and localize to antibody staining in IHC samples. Primary and secondary antibodies can be removed from the tissue and using antibody stripping after the Aluora dye signal development has completed without decreasing the intensity of the fluorophore deposited. Once antibodies are removed, the tissue can be reprobed with a primary antibody from the same or different host species (raised in rabbit or mouse) without risk of cross-reactivity, followed by detection with another round of Aluora spatial amplification. For added convenience and higher throughput, these steps can be performed on an automated slide stainer such as the Leica Bond RX systems.
Figure 2. Illustration of the cyclic staining process using Aluora spatial amplification. The protocol begins with antigen retrieval followed by blocking of nonspecific epitopes. The tissue can then be labeled with the primary antibody followed by the poly-HRP secondary reagent. Finally, the addition of the Invitrogen Aluora dye will covalently deposit the fluorophore onto the tissue. This process can be repeated for each primary antibody starting from Step 1 where the antibody complex will be removed.
Aluora spatial amplification reagents selection guide
| Aluora 430 Spatial Reagent | Aluora 488 Spatial Reagent | Aluora 514 Spatial Reagent | Aluora 555 Spatial Reagent | Aluora 594 Spatial Reagent | Aluora 647 Spatial Reagent | Aluora 700 Spatial Reagent | Aluora 750 Spatial Reagent* | |
| Detection | Fluorescence signal amplification of 100X | |||||||
| Ex/Em | 427/499 | 493/518 | 512/529 | 553/567 | 589/615 | 652/670 | 687/706 | 757/783 |
| Channel | 430 | 488 | 514 | 555 | 594 | 647 | 700 | 750 |
| EVOS filter | CFP | GFP | YFP | RFP | Texas Red | Cy5 | Cy5.5 | Cy7 |
| Sensitivity | Excellent | |||||||
| Application | Multiplex staining up to 8-plex for fluorescence spectral and spatial imaging applications | |||||||
| Automation | Compatible with multiplex staining on automated slide stainers such as Leica Bond | |||||||
| Individual Aluora reagents* | AS430HRP | AS488HRP | AS514HRP | AS555HRP | AS594HRP | AS647HRP | AS700HRP | AS750HRP |
| Aluora Goat anti-Mouse HRP Kits** | A40001329 | A40001330 | A40001331 | A40001332 | A40001333 | A40001334 | A40001335 | A40001336 |
| Aluora Goat anti-rabbit HRP Kits** | A40001337 | A40001338 | A40001339 | A40001340 | A40001341 | A40001342 | A40001343 | A40001344 |
| Aluora Streptavidin HRP Kits** | A40001345 | A40001346 | A40001347 | A40001348 | A40001349 | A40001350 | A40001351 | A40001352 |
| Aluora Spatial Rainbow Kit | A40002450 | |||||||
* The Aluora 750 Spatial Reagents are near IR tyramide-like reagents that covalently attach to tissue in a single step. Enzyme inactivation and antibody stripping can be immediately performed for subsequent signal amplification reactions, offering an advantage over the other near-IR signal amplification reagents on the market for spatial biology. ** Aluora spatial amplification kits contain all the necessary components to perform the assay. Multiplex experiments can be performed by purchasing additional Aluora spatial reagents. | ||||||||
New to spatial biology? We’re here to help. Get support for building your experiments here
Data image gallery from Aluora spatial amplification experiments
Figure 3. Invasive ductal carcinoma tissue stained with the Aluora Spatial Rainbow Kit and DAPI. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora Spatial Rainbow Kit (Cat. No. A40002450). Composite image with zoomed inset. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 4. Invasive ductal carcinoma tissue stained with the 8-plex Aluora spatial amplification assay and DAPI shown in individual tiles. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora Spatial Rainbow Kit (Cat. No. A40002450). Tiles represent individual Aluora dye colors. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 5. Single cell segmentation and phenotyping reveal spatial distribution of immune cell subpopulations. Human invasive ductal carcinoma of breast tissue analyzed to identify single cell target expression and specific localization of immune cell subpopulations. (A) Composite image using the Aluora Spatial Rainbow Kit (Cat. No. A40002450). (B) Tissue segmentation identifying vimentin positive and negative regions. (C) 0.7 mm2 area analyzed containing 10,454 cells, with white cell segments indicating dual positive proliferating & immune cell phenotypes. (D) Identification of region-specific cell localization. Analysis of the multiplex immunofluorescence stitched image was performed on the Indica Labs HALO (version 4.0.5107.318) software.
Figure 6. Invasive ductal carcinoma tissue stained with the 8-plex Aluora spatial amplification assay and DAPI. Human invasive ductal carcinoma of breast tissue processed and stained with the Aluora spatial amplification 8-plex assay. Composite image shown on the left and individual unmixed channels shown on the right. Images and spectral unmixing were performed on the EVOS S1000 Spatial Imaging System.
Figure 7. Aluora 594 Spatial Dye demonstrates excellent signal compared to Competitor O and secondary antibody equivalents with lower primary antibody concentrations. Human FFPE small intestine was incubated with 1:10,000 of PCNA Monoclonal Antibody (PC10) (Cat. No. 13-3900) then labeled with either Alexa Fluor 594 Secondary Antibody (Cat. No. A-11014), the reagents in the Aluora 594 Goat anti-Mouse HRP Kit (Cat. No. A40001331), or fluorophore equivalent of competitor 1. N=5 images analyzed for signal to background, P < 0.0001. Images were captured with an Invitrogen EVOS S1000 Spatial Imaging System.
Figure 8. Aluora spatial amplification reagents in human FFPE small intestine. Human FFPE small intestine was incubated with Mouse PCNA antibody (PC10, Cat. No. 13-3900) and labeled with Aluora 430 Goat anti-Mouse HRP Kit (Cat. No. A40001329) seen in green, Mouse Vimentin antibody (V9, Cat. No. MA5-11883) and labeled with Aluora 594 Goat anti-Mouse HRP Kit (Cat. No. A40001333) seen in red and Mouse SMA antibody (1A4, Cat. No. 14-9760-82) and labeled with Aluora 700 Goat anti-Mouse HRP Kit (Cat. No. A40001336) seen in white, and a DAPI nuclear stain. The slides were mounted in Prolong Glass Antifade Mountant. Images were captured with an Invitrogen EVOS M7000 Imaging System (see information about EVOS Imaging Systems).
Figure 9. Aluora spatial amplification reagents in human FFPE small intestine. Human FFPE small intestine was incubated with Mouse PCNA antibody (PC10, Cat. No. 13-3900) and labeled with Aluora 647 Goat anti-Mouse HRP Kit (Cat. No. A40001334) seen in red, Mouse Vimentin antibody (V9, Cat. No. MA5-11883) and detected with Aluora 488 Goat anti-Mouse HRP Kit (A40001330) seen in green and Mouse SMA antibody (1A4, Cat. No. 14-9760-82) and detected with Aluora 700 Goat anti-Mouse HRP Kit (Cat. No. A40001336) seen in white and a DAPI nuclear stain. The slides were mounted in Prolong Glass Antifade Mountant. Images were captured with an Invitrogen EVOS M7000 Imaging System (see information about EVOS Imaging Systems).
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Webinar: High-plex spatial proteomics workflow
Advancements in spatial proteomic workflows, featuring enzyme-activated spatial amplification reagents, have revolutionized cancer research capabilities. This webinar will include an explanation of techniques that allow for the simultaneous detection of up to eight biomarkers in tissues, driving multi-dimensional analysis of breast cancer specimens and valuable insights into intricate cellular interactions and phenotypes within tumor microenvironments.
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