Staining Neurological & Muscle Flashcards
(25 cards)
Neuropathology Techniques
- Neuropathology: H&E remains the most useful and commonly used stain
- Neuropathology : relies heavily on IHC as well as some special stain.
Neurological Tissue Components - CNS and PNS
CNS comprises the brain and spinal cord
PNS comprises motor and sensory neurons.
Neuropathological tissues comprises mainly neurons (cell body, dendrites, and axons) and supporting Glial cells/Neuroglia, as well as meninges and blood vessels.
Techniques for Neurons
Neurons - H&E: Yellow (oligodendrocytes), Orange: Astrocytes, Blue: Neurons.
Neurons - IHC
- Neurofilament protein (NFP) demonstrates intermediate filaments expressed in mature neurons
- PGP9.5 & NSE – cytoplasmic proteins both strongly expressed in neurons but not specific to nerve cells
- NeuN (Neuronal Nuclear Protein) - a neuron-specific DNA binding protein
- Synaptophysin - a membrane glycoprotein component of pre-synaptic neurosecretory vesicles; useful marker of neuroendocrine differentiation (also stains metastatic neuroendocrine tumours)
- Chromogranin A - a protein of the dense core matrix of neurosecretory vesicles; used predominantly to elucidate neuroendocrine differentiation in tumours
Techniques for axons and neuronal processes - Axons - Special stains
- Luxol Fast Blue - stains Myelin; useful for demyelinating diseases such as Multiple Sclerosis and Guillan-Barre Syndrome
- Toluidine blue - stains peripheral nerve
Techniques for Glial cells - What are Glial cells?
Supporting cells of the CNS including: Ependymal cells, astrocytes, oligodendrocytes, microglia
Axons - IHC
- Myelin Basic Protein (MBP) stains myelin sheaths.
- S100- used in diagnosis of tumours derived from Schwann cells.
Glial cells - IHC: GFAP
GFAP (Glial Fibrillary Acid Protein)
Can be used as a marker of brain injury
Can be used to assess whether a tumour is of glial origin
Glial cells - special stains
- Cresyl Violet - stains oligodendrocytes
Neurodegenerative Diseases - inclusion bodies
Many neurodegenerative diseases characterised by inclusion bodies (accumulations of proteins) that can be demonstrated using IHC.
Demonstration of neurodegeneration: Diseases, inclusion bodies and how they are demonstrated using IHC
Disease->Inclusion-> Demonstrated by
Alzheimer’s , Neurofibrillary tangles, Tau protein, ubiquitin/p62
Parkinson’s, Lewy bodies, alpha-synuclein, ubiquitin/p62
Motor Neuron Disease, Motor Neuron disease inclusion, Ubiquitin/p62, TDP-43
Multiple system atrophy, Glial cytoplasmic inclusion, alpha-synuclein, ubiquitin, p62
Picks disease, Pick body, Tau protein, ubiquitin/p62
How is Alzheimer’s disease visualised?
via its plaques and tangles using IHC.
Plaques – Beta Amyloid (IHC)
Tangles - Tau protein (IHC)
Parkinson’s Disease - Visualisation
Lewy bodies:
- H&E
- Alpha synuclein (IHC)
Picks disease - Visualisation
Pick bodies via Tau Protein using IHC
Muscle Biopsy samples
Skeletal muscle taken via biopsy needle
Frozen sections needed
Often assessing enzyme activity (disrupted by fixation)
Muscle Biopsy samples - staining techniques used
routine H&E is used for general morphology of muscle biopsies, but frequently other Cellular Pathology staining techniques are required:
Special stains
IHC
Enzyme histochemistry
Muscle biopsy samples ; Special stains used
- Gomori Trichrome: used to demonstrate abnormal mitochondrial aggregates, the rimmed vacuoles of inclusion body disorders, connective tissues, and ‘ragged red’ fibres suggestive of mitochondrial cytopathies)
- PAS (Periodic Acid Schiff) +/- Diastase: used to demonstrate glycogen in muscle tissues; accumulation of glycogen is indicative of glycogenoses
- Oil Red O/Sudan Black: used to demonstrate accumulation of lipids in muscles which can indicate carnitine deficiency
Muscle biopsy samples: IHC. What can IHC detect in myopathy (musclar disease)?
MHC (Major Histocompatibility Complex) class I proteins are overexpressed in some inflammatory - detectable by IHC
Muscle biopsy samples: IHC. What IHC stains are used to detect in myopathy (muscular disease)
Dystrophy-related IHC stains include Dystrophin 1, 2 and 3, sarcoglycans, dysferlin, merosin, caveolin, emerin and spectrin).
In normal muscle, Dystrophin is located beneath the cell membrane of muscle fibres.
Dystrophin: rod shape protein that links intracellular cytoskeleton network to transmembrane components of the DGC, including dystroglycan, sarcoglycans and sarcospan.
DCG: dystrophin complex - acts as a membrane stabilizer during muscle contraction to prevent contraction-induced damage
In Becker’s disease this staining is reduced, and in Duchenne muscular dystrophy the staining is absent
Enzyme Histochemistry
Branch of histochemistry using specific enzymatic reactions within tissues
Commonly used on muscle biopsies
Frozen sections are essential as the cross-linking nature of additive fixatives (e.g formalin) denatures enzymes of interest within muscle tissues.
ATPase staining
Assess fibre type
Alkaline pre-incubation demonstrates type 1 (slow) fibres
Acid pre-incubation demonstrates type 2 (fast fibres
NADH staining
Internal fibre architecture
Type 1 dark and Type 2 light
What does NADH staining target?
Targets fibres with vacuoles and loss of myofibrillar architecture (neurogenic process)
Amyotrophic? Lateral Sclerosis (ALS)
Atrophied fibres stain darkly
Hypertrophied fibres appear larger and paler
using alkaline phophatase..?
