Moran CORE | Stains and Processing

Stains and Processing

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Tissue Processing

Fixation in most cases is 10% neutral buffered formalin
For electron microscopy, fix tissue in glutaraldehyde
Fix in ethyl alcohol for cytologic preparations
For immunofluorescence, fix with Michel medium
Frozen sections and special stains (Oil Red O) require fresh tissue
- Can keep tissue fresh for 24 hours if pack with ice
- Place fresh tissue on saline-soaked gauze, not in dry gauze or container of saline
Embedding process removes water and replaces with paraffin
- Dissolves IOLs made of PMMA (polymethylmethacrylate), silicone, and haptics made of polypropylene
- Does not dissolve sutures
- Overnight process
4-6 μm sections of paraffin-embedded tissue are cut, affixed to a glass slide, and stained with various tissue dyes

Hematoxylin and Eosin (H&E)

H&E stain of cornea

Used in routine histopathologic staining
Blue: hematoxylin stains nucleus blue
Red: eosin stains cytoplasm red/pink
Does not stain lipidor mucin

Periodic Acid-Schiff (PAS)

PAS stain of corneal epithelium, Bowman’s membrane, and stroma

Used to stain basement membranes (Descemet’s membrane, lens capsule, epithelial basement membrane)
Magenta: glycogen and proteoglycans

Alcian Blue

Alcian Blue stain in Macular corneal stromal dystrophy

Used to stain mucopolysaccharides
- Useful in recognizing Macular corneal stromal dystrophy and cavernous optic atrophy of Schnabel
Blue: acid mucopolysaccharide

Colloidal Iron

Stain for mucopolysaccharides
- Helps with recognition of Macular corneal stromal dystrophy and cavernous optic atrophy of Schnabel
Blue: acid mucopolysaccharides
Red/purple: collagen

Alizarin Red

Calcification on explanted IOL

Used in band keratopathy and IOL calcification
Red: calcium

Von Kossa

Used to identify calcium, so useful in band keratopathy and IOL calcification
Less specific for calcium than Alizarin Red
Black: calcium phosphate salts

Congo Red

Congo red stain in Lattice corneal stromal dystrophy

Apple-green birefringence of corneal tissue

Used to stain amyloid
- Diagnoses amyloid deposits in all ocular tissues
- Also useful in Lattice corneal stromal dystrophy
Orange: amyloid
Shows apple-green birefringence under polarized light

Crystal Violet

Less commonly used to stain amyloid and used to recognize Lattice corneal stromal dystrophy
Purple/violet: amyloid

Thioflavin T

Stains for amyloid and used to recognize Lattice corneal stromal dystrophy in immunocytochemistry
Fluorescent yellow: amyloid

Masson’s Trichrome

Masson’s Trichrome stain in Granular corneal stromal dystrophy

Tri-color stain used to distinguish cells from connective tissue
- Also used to diagnose Granular corneal stromal dystrophy
Blue: collagen
Dark red/purple: nuclei
Red: muscle and cytoplasm

Gram Stain

Gram stain showing gram-positive bacteria

Used if question of bacterial infection
Specifically stains the thick peptidoglycan cell walls of certain types of bacteria
Blue: Gram-positive bacteria
Red: Gram-negative bacteria
Yellow: corneal stroma

Ziehl-Neelsen

Used to identify atypical mycobacteria
AKA Acid-fast stain
Red: acid-fast organisms

Gomori Methenamine Silver (GMS)

GMS stain showing yeast

Primarily used to identify fungal elements
Black: fungal elements
Green/blue: background

Gridley

Gridley stain showing Acanthamoeba cysts in the cornea

Primarily used in the diagnosis of acanthamoeba
Gray/silver: acanthamoeba cysts
Green: background/stroma

Prussian Blue

Prussian blue stain of cornea

Used to stain for iron in tissue, such as in hemosiderosis bulbio
Iron lines are left wherever tears pool and at elevations (keratoconus, pterygium)
Blue: iron

Verhoeff-van Gieson

Stains for elastin, which can be helpful in identifying the elastic layer of temporal artery
Black: elastic fibers

Oil Red O

Oil Red O stain showing arcus senilis of the cornea

Stains for lipid in frozen sections of fresh tissue
- Helps diagnose sebaceous carcinoma versus other epithelial-derived malignancies
Red: lipid

Sudan Black

Also stains for lipid in frozen sections of fresh tissue
- Helps diagnose sebaceous carcinoma versus other epithelial-derived malignancies
Used less often than Oil Red O
Black: lipid

Immunohistochemistry (IHC)

S-100 immunohistochemistry stain identifying melanocytes

How IHC works
- Cells present specific antigens
- Primary antibody binds cell antigen
- Secondary antibody binds to primary antibody
- Chromophore on secondary antibody gives color signal seen on microscopy
Common antigens and cell types in IHC
- Cytokeratins: cells of epithelial origin (adenoma, carcinoma)
- Desmin, myoglobin, actin: smooth and skeletal muscle (leiomyoma, rhabdomyosarcoma)
- S-100: neuroectoderm (schwannoma, neurofibroma, melanoma)
- HMB-45, Melan A: melanocytes (nevus, melanoma)
- Chromogranin, synaptophysin: neuroendocrine cells (metastatic carcinoid, small cell carcinoma)
- Leukocyte common antigen (CD45): hematopoietic cells (leukemia, lymphoma)
- CD antigens: white blood cell subtypes
- Her2/Neu, c-Kit: metastatic breast carcinoma prognosis and treatment

Electron Microscopy

TEM of Iris Pigment Epithelium Leiomyoma

Uses an electron beam rather than a beam of light (photons)
Electrons have a smaller wavelength, so can achieve a higher resolution
Specimen must be dehydrated and embedded in resin
Fix tissue with glutaraldehyde instead of formalin
Stain with heavy metal (lead, uranium, tungsten) to scatter electrons and create contrast in different structures
Needs high-vacuum environment to allow electron beam to be straight
Generates black-and-white images
- Black: electron dense
Transmission electron microscopy (TEM)
- Creates cross-section image of specimen
- Requires very thin sections that are 100 nm thick
Scanning electron microscopy (SEM)
- Creates image of the specimen’s surface
- Electrons do not pass through specimen, so thin sections not needed
- Lower voltage electron beam than TEM
- Can use low-vacuum environment
- Coat with gold to prevent charging the specimen from the electron beam and increase the signal-to-noise ratio
Lengthy specimen preparation and very expensive equipment, therefore very rarely used in ocular pathology

Summary of Common Stains in Ocular Pathology

Stains	Colors	Examples
H&E	Blue: nucleus Red/pink: cytoplasm	Routine tissue stain
PAS	Magenta: basement membrane glycogen and proteoglycans	Descemet membrane
Alcian Blue	Blue: acid mucopolysaccharides	Macular corneal stromal dystrophy
Colloidal Iron	Blue: acid mucopolysaccharides Red/purple: collagen	Macular corneal stromal dystrophy
Alizarin Red	Red: calcium	Band keratopathy
Von Kossa	Black: calcium phosphate	Band keratopathy
Congo Red	Orange with red-green birefringence: amyloid	Lattice corneal stromal dystrophy
Crystal Violet	Purple/violet: amyloid	Lattice corneal stromal dystrophy
Thioflavin T	Fluorescent yellow: amyloid	Lattice corneal stromal dystrophy
Masson’s Trichrome	Blue: collagen Dark red/purple: nuclei Red: cytoplasm, muscle	Granular corneal stromal dystrophy
Gram Stain	Blue: Gram-positive bacteria Red: Gram-negative bacteria Yellow: corneal stroma	Bacterial infections
Ziehl-Neelsen	Red: acid-fast organisms Blue: background	Tuberculosis
GMS	Black: fungal elements Blue-green: background	Fungal infections
Gridley	Gray-silver: acanthamoeba cysts Green: background	Acanthamoeba infections
Prussian Blue	Blue: iron	Hemosiderosis bulbi
Verhoeff-van Gieson	Black: elastic fibers	Temporal artery biopsy
Oil Red-O	Red: lipid	Sebaceous carcinoma
Sudan Black	Black: lipid	Sebaceous carcinoma

References

Harper RA. Basic Ophthalmology. Amer Academy of Ophthalmology; 2010.

Yanoff M, Fine BS. Ocular Pathology, A Text and Atlas. Lippincott Williams & Wilkins; 1989.