Multiplex Immunofluorescence – Tissue Staining & Quantitative Image Analysis Services

Yes. For fluorescent stains, especially in muscle, CNS, and human tissues where autofluorescence can be problematic, we routinely apply quenchers such as Sudan Black B, TrueBlack, or TrueView quenching solutions. These steps substantially improve signal-to-noise ratios while preserving antigenicity and fluorophore integrity. 

Yes. We can incorporate amyloid- and aggregate-specific dyes into multiplex panels when compatible with the tissue and fixation conditions. Thioflavin S is commonly used for β-sheet amyloid detection, while pFTAA offers higher sensitivity and can resolve conformational differences between protein fibrils. Other aggregation dyes, such as  AmyTracker, can also be included upon request.

Our standard workflow supports 4-5 markers plus DAPI in a single staining round, typically using fluorophores such as AF488, AF555, AF647, and AF750. These dyes are selected to provide high brightness, strong photostability, and minimal spectral overlap. We routinely combine primary antibodies from multiple host species and maintain a broad portfolio of validated antibodies for multiplex use. When appropriate, we also incorporate pre-conjugated primary antibodies to further expand panel flexibility and reduce cross-reactivity.

Yes. We provide full support for panel design. These services include selecting antibodies based on host species, avoiding cross-reactivity, choosing fluorophores with appropriate spectral spacing, and defining antigen retrieval conditions compatible with all targets. Panels are validated on positive and negative controls to ensure specificity before being applied to study samples. 

Yes. We offer quantitative analysis ranging from standard intensity measurements to highly advanced image analytics. Our pipeline includes colocalization assessment, cell and fiber segmentation, regional quantification, and detailed NMJ morphometric profiling. We also analyze the local tissue microenvironment, including glial and inflammatory landscapes, microglial activation states, cellular proximity relationships, and spatial mapping of pathological markers within their surrounding structures. Results can be delivered as raw data, fully processed tables, or publication-ready figures according to your project needs. 

We do. Many projects require antibodies that are not part of our standard panel, and we frequently validate new primary antibodies, rare antigens, or non-standard species. Custom assays can be designed entirely around your biological question and tissue type, including low-abundance markers, conformational epitopes, or specialized intracellular targets. 

Yes. All antibodies undergo validation using positive and negative control tissues as well as omission controls. Secondary antibodies are chosen to avoid cross-species binding, and for mouse primaries on mouse tissue we use appropriate mouse-on-mouse kits and blocking strategies to prevent background arising from endogenous IgG. Dilution series and signal optimization are performed when needed to achieve clean, high-contrast staining. 

We routinely work with mouse, rat, and human tissues, including fixed-frozen and FFPE samples. Our workflow supports a wide range of tissue types, including muscle, CNS, PNS, tumors, and organoids. For FFPE samples, we typically handle 3 µm sections, while fixed-frozen samples are often prepared as thicker sections between 20 and 40 µm. In these thicker tissues, we apply extended antibody incubations and optimized permeabilization protocols to achieve deep, uniform staining throughout the section. 

Mouse primary antibodies on mouse tissue require specialized handling. We use dedicated M.O.M. fluorescence kits, Fc receptor blocking, and serum or IgG blocking solutions (typically goat or donkey serum depending on the secondary species). These measures significantly reduce background and greatly improve the signal-to-noise ratio. 

Our imaging is performed on high-resolution automated widefield fluorescence systems optimized for multiplex work. These platforms support in focus whole-slide imaging and high dynamic range acquisition.

Simply contact our team with a short description of your targets, tissue type, and goals. We will schedule a consultation, design or refine your panel, establish feasibility, and then prepare a detailed quote and project timeline. 

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Alexa Fluor Dyes (AF488, AF555, AF647, AF750): a set of highly photostable fluorophores enabling four-color multiplexing on a single slide. These dyes provide high signal-to-noise ratios, minimal spectral overlap, and allow quantitative, spatially resolved co-localization analysis of multiple proteins simultaneously. 

Antigen Retrieval (HIER, enzymatic, formic acid): methods that unmask epitopes in FFPE tissue to enable high-affinity binding of multiple fluorescently labeled antibodies. Critical for targets including TDP-43, pTDP-43, Tau, pSyn129, Aβ, MBP, MOG, Olig2, CC1, Iba1, and GFAP. Optimized antigen retrieval enhances multiplex signal preservation and minimizes cross-channel interference. 

Automated mIF Staining: high-throughput, standardized fluorescence staining optimized for multi-cohort preclinical studies. Allows simultaneous detection of multiple targets on a single tissue section with reproducible signal intensity, reducing variability compared to sequential IHC. Primary antibodies from different species are used to allow distinct detection, and species-specific fluorescent secondary antibodies bind selectively to each primary, enabling accurate multiplexing. 

Co-localization Analysis: a quantitative approach to measure spatial overlap of fluorescently labeled proteins within a single tissue section. Enables determination of whether specific proteins are present in particular cell types, assessment of relative protein distribution, and mapping of neuron–glia adjacency. Focuses on molecular-level co-occurrence rather than broader tissue architecture. 

DAPI Counterstain: a blue fluorescent nuclear dye used in mIF for automated nuclear segmentation, cell identification, and morphometric quantification across CNS regions. Provides a stable reference for tissue architecture and cellular localization in multi-channel fluorescence imaging. 

FFPE Tissue mIF: a standardized, archival-ready format enabling robust multi-color fluorescence staining, long-term reproducibility, and retrospective analysis of neurodegenerative disease cohorts. mIF allows assessment of multiple markers on the same tissue section, unlike chromogenic IHC which is generally single-target per slide. 

Fluid-to-Tissue and Imaging Correlation Including MRI: integration of mIF with translational biomarkers such as NF-L, GFAP, Aβ, and pTau, combined with MRI-derived measures (atrophy, T2*, diffusion tensor imaging, lesion load). This multimodal approach enables spatially resolved correlation between cellular pathology and functional or structural imaging endpoints. 

Fluorescent Detection: use of fluorophore-conjugated secondary antibodies that specifically bind to the primary antibodies, providing high-intensity, quantitative signals. Fluorescence allows detection of low-abundance proteins and simultaneous analysis of multiple markers on the same section, preserving spatial and morphological context. 

Motor Neuron and NMJ mIF Markers: fluorescent markers including ChAT, NeuN, βIII-tubulin, SV2, and α-bungarotoxin (BTX) for postsynaptic acetylcholine receptors. Enables identification of motor neurons, distal axons, and NMJ morphology with subcellular resolution, supporting multi-marker co-localization studies and detailed mapping of synaptic integrity not achievable with traditional IHC. 

Multiplex Immunofluorescence (mIF): a technique enabling simultaneous detection of multiple protein targets in the same tissue section using distinct fluorophores. Preserves spatial relationships and tissue morphology, allowing detailed mapping of cellular co-expression, cell–cell interactions, and microenvironment organization at single-slide resolution. 

Myelin and Axonal Pathology mIF: markers such as MBP, MOG, NF-L, APP, Olig2, and CC1 conjugated to distinct fluorophores. Facilitates simultaneous quantification of demyelination, oligodendrocyte lineage states, and axonal injury on the same tissue section, preserving spatial relationships. 

Neurodegenerative pathology mIF Markers: fluorescent detection of TDP-43, pTDP-43, Tau, pTau, Aβ, and pSyn129. Supports co-localization studies of protein aggregates, enabling assessment of overlapping pathology and subcellular distribution within neurons and glia. 

Neuroinflammation mIF Markers: fluorescent markers including Iba1, GFAP, CD68, CD3, and CD45. Allows single-slide assessment of microglial activation, astrogliosis, and immune infiltration, revealing spatial relationships between immune cells and neurons, which is challenging in single-target IHC. 

Quantitative mIF Image Analysis: automated extraction of fluorescence intensity, protein co-localization, cellular density, morphological metrics, and spatial distribution using high-end digital pathology pipelines. Multi-marker quantification enables direct comparison of pathological processes on the same section. 

Spatial and Morphological mIF Analysis: high-resolution characterization of tissue architecture and microenvironment organization. Enables direct visualization of multiple cellular components and their spatial relationships, including glial activation and inflammatory responses surrounding pathological features such as amyloid plaques. Highlights regional pathology, cellular clustering, and multicellular interactions at the tissue scale, providing insights not accessible with conventional IHC. 

Therapeutic Efficacy mIF Readouts: quantitative measurement of treatment-induced changes in multi-marker expression, co-localization, cellular responses, and preservation of tissue structure. Single-slide multiplexing enables detection of subtle therapeutic effects across neuronal, glial, and synaptic populations while maintaining spatial and morphological context. 

Whole-Slide Fluorescence Scanning: high-resolution, multi-channel imaging of entire tissue sections. Captures spatial distribution of multiple fluorescent markers while preserving tissue architecture, enabling comprehensive quantitative analysis and detailed mapping of cellular and microenvironmental organization across large cohorts.