Immunohistochemistry (IHC) – Tissue Staining & Quantitative Image Analysis Services

We use a counterstain, called Acid Blue 129, that our team developed several years ago (Zehntner, 2008). This stain has a homogenous light blue background that provides excellent contrast to the red-brown chromogen and is well-suited for image segmentation and quantitative analysis.

We typically use AEC for our IHC studies. This chromogen provides a crisp, strong red-brown color and does not have the toxicity associated with DAB.

The antigen retrieval method really depends on the particular stain/antibody. For example, for phosphorylated α-synuclein, we use proteinase K pre-treatment. For β-amyloid, we use formic acid pre-treatment. For many stains, we use heat-induced epitope retrieval (HIER) using various buffers.

Yes. Our R&D team routinely validates new antibodies, optimizing dilution, retrieval, and detection conditions to ensure robust, reproducible staining.

Absolutely. All quantitative analyses can be conducted under blinded conditions upon request to minimize bias and ensure data integrity.

Yes, we can perform triple staining: amyloid-β plaques can be visualized with DAB (brown), microhemorrhages with Prussian blue (blue), and tissue counterstained with eosin (pink) for contrast. 

Both MC1 and AT8 antibodies bind Tau protein; however, MC1 recognizes a conformation-specific, misfolded form of Tau, whereas AT8 detects phosphorylation-specific epitopes associated with hyperphosphorylated Tau. 

The choice of markers depends on your biological question, tissue type, and animal model. We can use well-established markers or work with your custom antibodies. Examples of commonly used markers include: 

• Gliosis / Inflammation: GFAP (astrocytes), Iba1 (microglia/macrophages) 

• Neuronal Health / Loss: NeuN (neuronal nuclei) 

• Protein Aggregation / Pathology: pSyn (phosphorylated α-synuclein), pTDP-43, AT8, MC1, amyloid-beta 

Mebratie, D. Y., Dagnaw, G. G. Review of immunohistochemistry techniques: Applications, current status, and future perspectives. Semin. Diagn. Pathol., 41: 154–160, 2024; doi: 10.1053/j.semdp.2024.05.001

Velasco-Vales, V., Soria-Céspedes, D., Cuesta-Mejías, T. C., & Padrón-Pérez, N. C. Immunohistochemistry, Quality Control, and Principles of Validation in the Central Nervous System. Methods Mol. Biol., 2422: 203–216, 2022; doi: 10.1007/978-1-0716-1948-3_14

Zehntner, S. P., Chakravarty, M. M., Bolovan, R. J., Chan, C., & Bedell, B. J. Synergistic tissue counterstaining and image segmentation techniques for accurate, quantitative immunohistochemistry. J. Histochem. Cytochem., 56: 873–880, 2008; doi: 10.1369/jhc.2008.950345

Acid Blue 129 Counterstain: a light blue nuclear and background stain optimized for automated segmentation, cell identification, and morphometric quantification across CNS regions. 

Antigen Retrieval (HIER, enzymatic, formic acid): a process that restores masked epitopes in FFPE tissue to ensure high-affinity antibody binding. Common targets include TDP-43, pTDP-43, Tau, pSyn129, Aβ, MBP, MOG, Olig2, CC1, Iba1, and GFAP. 

Automated IHC Staining: high-throughput, standardized chromogenic staining optimized for multi-cohort preclinical studies. It ensures reproducible detection of disease-relevant proteins across large sample sets. 

Chromogenic Detection (AEC, DAB): a method that produces stable, permanent colorimetric signals ideal for whole-slide brightfield imaging, quantitative analysis, and longitudinal comparison of pathological burden. 

FFPE Tissue IHC: a standardized, archival-ready format that enables robust staining performance, long-term study reproducibility, and retrospective comparison across studies and therapeutic cohorts. 

Fluid-to-Tissue and Imaging Correlation Including MRI: integration of IHC with translational biomarkers such as NF-L, GFAP, Aβ, and pTau, along with MRI-derived measures including atrophy, T2*, diffusion tensor imaging (DTI), and lesion load. This approach enables multimodal assessment of disease progression and therapeutic impact. 

Immunohistochemistry (IHC): a technique that detects and spatially localizes specific proteins in formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissue sections using chromogenic reporters.

Motor Neuron and Neuronal IHC Markers: targets including ChAT, NeuN, βIII-tubulin, SV2, and synaptic proteins for identifying neuronal populations and quantifying neuronal and synaptic degeneration. 

Myelin and Axonal Pathology IHC: markers such as MBP, MOG, NF-L, APP, Olig2, and CC1 for quantifying demyelination, oligodendrocyte lineage states, and axonal injury or loss. 

Neurodegeneration IHC Markers: chromogenic detection of TDP-43, pTDP-43, Tau, pTau, Aβ, and pSyn129 for assessing hallmark pathological processes and progressive CNS degeneration. 

Neuroinflammation IHC Markers: markers including Iba1, GFAP, CD68, CD3, and CD45 for profiling microglial activation, astrogliosis, and inflammatory infiltration in the brain and spinal cord. 

Quantitative IHC Image Analysis: automated quantification of protein expression levels, cellular density, morphological metrics, and spatial distribution patterns using validated digital pathology pipelines.

Spatial and Morphological IHC Analysis: characterization of tissue architecture, microenvironment organization, glial-neuronal interactions, and region-specific pathological features relevant to neurodegenerative disease progression. 

Therapeutic Efficacy IHC Readouts: quantitative measurement of treatment-induced changes in protein expression, pathological burden, cellular responses, tissue integrity, and neurodegenerative or inflammatory signatures. These readouts provide robust, mechanistically aligned endpoints for preclinical drug evaluation. 

Whole-Slide Brightfield Scanning: high-resolution digitization of entire tissue sections.