Stains and immunohistochemistry (IHC) are the workhorse tools of the neuropathology bench. Routine histology with hematoxylin and eosin (H&E) reveals broad patterns; a focused panel of special stains and IHC then refines the diagnosis — identifying the cell types involved, the proteins deposited, the infectious organisms present, and the molecular alterations relevant to classification and treatment. Modern neuropathology cannot be practiced without IHC, and the ability to read a stain panel intelligently is among the most useful clinical skills for the neurologist who works closely with the pathology service. This page covers the major stains and antibodies used in neuropathology, what they show, and the contexts in which they are most informative.

Special Stains

Hematoxylin and Eosin (H&E)

The default stain. Hematoxylin stains nuclei blue-purple; eosin stains cytoplasm and extracellular matrix pink-red. Almost every diagnostic conversation starts with H&E. Recognizable on H&E:

  • Cellular density and architecture.
  • Inflammation.
  • Necrosis.
  • Acute neuronal injury (red neurons).
  • Reactive astrocytes (gross morphology).
  • Microglial nodules.
  • Many inclusions (Lewy bodies, neurofibrillary tangles, plaques) — often visible but more clearly demonstrated by IHC.
  • Vascular changes.

Luxol Fast Blue (LFB)

Stains myelin a deep blue. Often combined with H&E or PAS to highlight myelin loss against nuclear and cytoplasmic detail. Key uses:

  • Identification of demyelination (MS plaques, ADEM, PML).
  • White matter pallor (chronic small vessel disease, leukodystrophies).
  • Definition of plaque borders.
  • Distinguishing white from gray matter in sectioning.

Bielschowsky Silver Stain

Impregnates with silver, identifying axons and neurofibrillary tangles. Classic for:

  • Alzheimer disease: plaques and tangles.
  • Axonal preservation versus loss (assessment of demyelinated plaques).
  • Axonal pathology in any disease.

Other Silver Stains

  • Bodian: similar to Bielschowsky for axons and tangles.
  • Holmes: axons.
  • Gallyas: tau-containing inclusions (often used to highlight tangles and Pick bodies).

Periodic Acid-Schiff (PAS)

Stains carbohydrates and glycoproteins magenta. Identifies:

  • Fungi (e.g., Aspergillus hyphae, Candida).
  • Lafora bodies (intracellular polyglucosan in Lafora disease).
  • Corpora amylacea.
  • Basement membranes.
  • Glycogen.

Gomori Trichrome

Multi-color stain for muscle biopsy. Distinguishes:

  • Type 1 fibers (red) from type 2 fibers (paler).
  • Endomysial connective tissue (blue-green).
  • Mitochondrial accumulations (ragged-red fibers in mitochondrial myopathies).
  • Nemaline rods (in nemaline myopathy).

Modified Gomori Trichrome

Specifically for muscle biopsy; classical “ragged-red fiber” appearance of mitochondrial proliferation under the sarcolemma.

NADH-Tetrazolium Reductase

Muscle histochemistry. Highlights mitochondrial distribution and structural abnormalities (target fibers, central cores).

Cytochrome c Oxidase (COX) and Succinate Dehydrogenase (SDH)

Muscle histochemistry for mitochondrial disease. COX-negative fibers with SDH-positive fibers (the “blue ragged fibers” pattern) indicate mitochondrial DNA mutations.

Congo Red

Stains amyloid red on bright-field; apple-green birefringence under polarized light is pathognomonic. Used in:

  • Cerebral amyloid angiopathy (vessel walls).
  • Amyloid plaques in AD (less specific than Aβ IHC now).
  • Systemic amyloidosis with CNS or PNS involvement.
  • Amyloid neuropathies (peripheral nerve biopsy).

Thioflavin S or T

Fluorescent staining of amyloid. Highlights senile plaques and neurofibrillary tangles in AD.

Prussian Blue (Perls)

Identifies iron (hemosiderin) as blue. Demonstrates:

  • Old hemorrhage.
  • Hemosiderin deposition in cerebral amyloid angiopathy with microbleeds.
  • Brain iron accumulation in NBIA (eye of the tiger pattern with PANK2 mutation).

Acid-Fast Stains (Ziehl-Neelsen, Fite)

Identifies mycobacteria (TB, leprosy). Important in:

  • Tuberculous meningitis.
  • Tuberculomas.
  • Spinal cord granulomas.

Gram Stain

Bacterial identification in meningitis, abscess, encephalitis.

Methenamine Silver (GMS)

Stains fungi black against pale background. Identifies:

  • Cryptococcus.
  • Aspergillus.
  • Mucor.
  • Pneumocystis.

Giemsa

Useful for protozoan organisms (toxoplasma, malaria) and intracellular inclusions.

Cresyl Violet (Nissl Stain)

Stains Nissl substance (rough ER) of neurons. Useful for assessing neuronal cytology, especially neuronal loss and chromatolysis.

Immunohistochemistry in Neuropathology

Glial Cell Markers

  • GFAP (Glial Fibrillary Acidic Protein): astrocytes. Stains reactive astrocytes intensely; also stains astrocytic tumors (astrocytoma, glioblastoma) and ependymal cells.
  • S100: astrocytes, Schwann cells, melanocytes, and some glial tumors.
  • SOX10: oligodendroglial lineage cells and Schwann cells. Useful in distinguishing oligodendroglioma and identifying schwannomas.
  • Olig2: oligodendroglial lineage; nuclear staining in oligodendrogliomas and many astrocytomas.
  • CNPase: oligodendrocytes; less commonly used.

Neuronal Markers

  • Synaptophysin: synaptic vesicle protein. Stains neurons and neuroendocrine cells; widely used for neural tumors and synaptic density.
  • Chromogranin A: neuroendocrine granules.
  • NeuN (Neuronal Nuclei): mature neurons; nuclear staining. Useful for assessing neuronal density and identifying neuronal tumors (ganglioglioma).
  • Neurofilament (NF): neuronal cytoskeleton; identifies axons and neurons.
  • MAP2: dendritic marker.

Microglial / Macrophage Markers

  • CD68: macrophages, microglia (especially activated). Lysosomal marker; increases with activation.
  • IBA1 (AIF1): microglia constitutively; sensitive marker of resting and activated microglia.
  • HLA-DR: activated microglia.

Inflammatory Cell Markers

  • CD3: pan-T cell.
  • CD4, CD8: T-helper, T-cytotoxic subsets.
  • CD20: B cells.
  • CD45 (LCA): pan-leukocyte.
  • CD138: plasma cells.
  • Mac387: recently recruited macrophages (vs resident microglia).

Vascular Markers

  • CD31: endothelial cells. Highlights vascular density and microvascular proliferation (key feature of glioblastoma).
  • CD34: endothelium, hematopoietic precursors, and dermal/CNS spindle cells. Positive in some low-grade gliomas (DNET, PXA).
  • SMA: smooth muscle in vessel walls.

Proliferation Markers

  • Ki-67 (MIB-1): nuclear marker of proliferation. Quantified as percentage of cells positive — the “Ki-67 index” is a key component of tumor grading.
  • Phospho-histone H3 (pHH3): marks mitoses.

Tumor Markers

  • EMA: meningiomas (membrane), ependymomas (dot-like cytoplasmic), epithelial tumors. Distinguishing dot positivity is helpful for ependymoma.
  • Vimentin: mesenchymal cells; non-specific but useful for some tumors.
  • Cytokeratins (CKs): epithelial tumors (metastases, choroid plexus, craniopharyngioma).
  • S100, SOX10: schwannoma (positive both).
  • HMB-45, Melan-A: melanocytic tumors.
  • Inhibin: hemangioblastoma.
  • Beta-catenin: nuclear staining in WNT-activated medulloblastoma and adamantinomatous craniopharyngioma.
  • OCT3/4, SALL4: germ cell tumors.
  • SOX2: stem cell marker; some embryonal tumors.

Molecular Surrogate Markers (IHC for Genotype)

  • IDH1 R132H: the most common IDH1 mutation in adult diffuse glioma. Antibody-detectable. If positive, indicates IDH-mutant tumor; if negative in patient under 55 with diffuse glioma, sequencing for less common IDH mutations is required.
  • ATRX: nuclear loss indicates ATRX mutation (associated with diffuse astrocytomas, IDH-mutant).
  • p53: strong diffuse nuclear positivity suggests TP53 mutation (IDH-mutant astrocytomas).
  • H3 K27M: mutation-specific antibody; positive in diffuse midline glioma, H3 K27-altered (pediatric).
  • H3K27me3: loss of trimethylation; useful in H3 K27-altered tumors and posterior fossa Group A ependymomas.
  • H3 G34R/V: mutation-specific; pediatric high-grade glioma.
  • BRAF V600E: mutation-specific; PXA, ganglioglioma, some pilocytic astrocytomas, Langerhans cell histiocytosis, others.
  • L1CAM: posterior fossa Group A ependymoma.
  • YAP1: supratentorial ependymoma (YAP1 fusion subtype).
  • GAB1, YAP1: SHH medulloblastoma.
  • Filamin A: GTF2I-mutant or NAB2-STAT6 fusion (some tumors).
  • STAT6: solitary fibrous tumor / hemangiopericytoma (nuclear).
  • INI1 (SMARCB1): loss in atypical teratoid/rhabdoid tumor (AT/RT), some other rhabdoid tumors.
  • BRG1 (SMARCA4): loss in some AT/RT cases.

Neurodegenerative Disease Markers

  • Beta-amyloid (Aβ): stains amyloid plaques and CAA. Antibodies against Aβ40 and Aβ42 distinguish CAA-predominant vs plaque-predominant deposition.
  • Tau (AT8, AT100, MC-1, PHF-1): detect hyperphosphorylated tau in neurofibrillary tangles (AD), Pick bodies, glial inclusions of PSP/CBD, etc.
  • 3R and 4R tau-specific antibodies: distinguish Pick disease (3R) from PSP/CBD (4R) from AD (mixed 3R+4R).
  • Alpha-synuclein: Lewy bodies, Lewy neurites (PD, DLB), glial cytoplasmic inclusions of MSA. The pS129 phospho-specific antibody is more sensitive.
  • TDP-43 (and phospho-TDP-43): cytoplasmic inclusions in ALS, FTLD-TDP. Phospho-TDP-43 is highly specific.
  • FUS: FTLD-FUS subset and some ALS cases.
  • Ubiquitin, p62: broader inclusion markers; positive in many neurodegenerative inclusions.
  • Prion protein (3F4, KG9): prion disease.
  • Polyglutamine-specific (1C2): Huntington and some SCAs.

Infectious Disease Markers

  • HSV-1, HSV-2: herpes simplex encephalitis.
  • VZV: varicella zoster.
  • CMV: cytomegalovirus encephalitis.
  • HIV p24: HIV-infected multinucleated giant cells.
  • JC virus large T antigen, VP1: PML.
  • SV40 (cross-reactive with JC and BK): PML.
  • EBV LMP1, EBER (in situ): EBV-associated lymphomas, PCNSL.
  • Treponema pallidum: neurosyphilis.
  • Toxoplasma gondii: identifies organisms in toxoplasmosis.
  • West Nile virus: when available.

Routine Stain Panels by Diagnostic Question

Adult Glioma

  • H&E.
  • GFAP, OLIG2, SOX10.
  • IDH1 R132H, ATRX, p53.
  • Ki-67.
  • If IDH negative and patient < 55: IDH sequencing.
  • If glioblastoma: MGMT promoter methylation, EGFR amplification, 1p/19q if oligodendrogliomatous.
  • If oligodendroglioma: 1p/19q FISH or sequencing.

Suspected Meningioma

  • H&E.
  • EMA, vimentin.
  • Progesterone receptor.
  • SSTR2A (somatostatin receptor 2A) — sensitive meningioma marker.
  • Ki-67.
  • If atypical: TERT promoter mutation status.

Suspected Lymphoma

  • H&E.
  • CD20, CD3, CD45.
  • BCL6, MUM1 (DLBCL subtyping).
  • EBER in situ if EBV-associated.
  • MYC, BCL2 (double-hit lymphoma).

Demyelinating Lesion

  • H&E.
  • LFB / PAS for myelin.
  • Bielschowsky for axons.
  • CD68 for foamy macrophages.
  • CD3, CD20 for inflammation.
  • JC virus IHC if PML suspected.

Suspected Neurodegenerative Disease

  • H&E.
  • Disease-specific: Aβ, tau (3R, 4R, AT8), alpha-synuclein, TDP-43.
  • Ubiquitin, p62.
  • Prion protein if prion considered.

Muscle Biopsy

  • H&E.
  • Gomori trichrome.
  • NADH-TR (mitochondrial pattern, fiber type).
  • ATPase reactions (fiber type at various pH).
  • COX, SDH (mitochondrial).
  • Periodic Acid-Schiff (glycogen).
  • Oil Red O (lipid).
  • Dystrophin, sarcoglycans, dysferlin, caveolin-3, alpha-dystroglycan (dystrophy panel).
  • Major histocompatibility complex class I (MHC-I) — sarcolemmal expression in inflammatory myopathies.
  • CD3, CD20, CD8, CD68 — inflammation.
  • p62, TDP-43, LC3 — inclusion body myositis.

Nerve Biopsy

  • H&E.
  • Toluidine blue (semithin sections).
  • Electron microscopy.
  • Teased fiber preparations (axonal vs demyelinating).
  • Congo red (amyloid).
  • S100 (Schwann cells).
  • NF (axons).
  • CD3, CD20, CD68 (inflammation).

🔍 Did You Know?

Immunohistochemistry for the IDH1 R132H mutation — a single-amino-acid substitution in isocitrate dehydrogenase 1 — has transformed the workflow of adult diffuse glioma diagnosis. The mutation is present in approximately 90% of grade 2 and 3 astrocytomas and oligodendrogliomas in adults, and in a smaller fraction of glioblastomas (about 5-10% — the “secondary” glioblastomas, which biologically behave more like IDH-mutant astrocytomas). The R132H antibody is highly specific and produces strong cytoplasmic positivity in tumor cells. The clinical importance is that an IDH-mutant adult diffuse glioma carries a fundamentally different prognosis from an IDH-wildtype glioma — IDH-mutant tumors progress more slowly, respond better to chemotherapy and radiation, and are now treated with IDH-targeted agents such as vorasidenib. In a young or middle-aged adult with a diffuse glioma, a positive IDH1 R132H stain reframes the whole prognostic conversation. A negative IDH1 R132H stain in a patient under 55 prompts sequencing for less common IDH mutations (other IDH1 codons, IDH2 mutations) before settling on an IDH-wildtype diagnosis. The 2021 WHO classification embeds this molecular categorization at the core of adult glioma diagnosis — and a simple immunostain is the entry point.

Pitfalls and Pearls

  • Read the stain panel intelligently. Each stain answers a specific question; the combination is the diagnosis.
  • GFAP: astrocytes; positive in reactive gliosis and astrocytic tumors.
  • Synaptophysin + chromogranin + NeuN: neuronal lineage.
  • Ki-67 index: proliferation; critical for tumor grading.
  • IDH1 R132H positivity in adult diffuse glioma points to IDH-mutant tumor and changes management.
  • H3 K27M IHC: diffuse midline glioma, H3 K27-altered.
  • BRAF V600E: PXA, ganglioglioma, some pilocytic.
  • INI1 loss: atypical teratoid/rhabdoid tumor.
  • Congo red apple-green birefringence: amyloid pathognomonic.
  • LFB + Bielschowsky combination: distinguishes primary demyelination (LFB pale, axons preserved) from axonal damage (both lost).
  • Tau 3R vs 4R isoform IHC: distinguishes Pick disease (3R), PSP/CBD (4R), AD (mixed).
  • Phospho-TDP-43: highly specific marker of FTLD-TDP and ALS pathology.
  • Alpha-synuclein: Lewy bodies (PD, DLB), GCIs (MSA).
  • Modified Gomori trichrome: ragged-red fibers in mitochondrial disease.
  • MHC-I sarcolemmal expression: inflammatory myopathy.
  • Congo red on nerve biopsy: amyloid neuropathy (familial hATTR, AL).

References

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  3. Louis DN, Perry A, Wesseling P, et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol. 2021;23(8):1231-1251.
  4. Dubowitz V, Sewry CA, Oldfors A. Muscle Biopsy: A Practical Approach. 5th ed. Elsevier; 2020.
  5. Capper D, Zentgraf H, Balss J, Hartmann C, von Deimling A. Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathol. 2009;118(5):599-601.
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