NERVE
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| MALLORY PTAH | demonstrate glial fibers | Tungsten (20:1) in staining solution binds all available hematein to make BLUE LAKE = blue color to selected tissue components red-brown/salmon colored (neurons) stained by phosphotungstic acid | 10%NBF 6-8 microns | Cerebral Cortex tissue for glial fibers | PTAH solution (allow to ripen naturally, can use KMnO4), Lugol Iodine, Sodium thiosulfate, KMNO4, Oxalic Acid Mordant in ZENKERS/AA; Rinse; LUGOL IODINE; Decolor 95%ROH; Rinse, KMnO4;Wash, decolor in OXALIC ACID; Wash, stain in PTAH; Dehydrate,clear, mou | GLIAL, NUCLEI, MYELIN = Blue NEURONS = salmon | Holzer stain is better for glial fibers
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| CAJAL | demonstrate astrocytes (replaced by IHC) | Selective staining w/ Cajal gold sublimate | Formalin ammonium bromide, 2>x<25 days; wash if originally in 10%NBF FROZEN SECTIONS, 20-30u; free float sections | Section of cerebral cortex for astroctyes | Free-floating sections wash in DIh2o; transfer to GOLD SUBLIMATE in DARK; wash, treat w/ 5% SODIUM thiosulfate; wash, mount sections on slides, blot, dehydrate, clear, mount | Astrocyte w/ perivascular feet = black | use brown gold chlorid; not too long of fixation;
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| WEIL | demosntrate myelin to see if it's been broken down as axon degenerates | Mordant-hematox attaches to phospholipid component of myelin; regressive w/ 2 differentiations: ferric ammonium sulfate (excess mordant) & borax ferricyanide (microscopic, oxidizes); only myelin sheath and RBC left stained | 10%NBF; 20-15u paraffin sections | section of spinal cord or medulla | Incubate in stain solution (4% Ferric Ammonium Sulfate, Alcoholic hematoxylin,10%), H2O); wash, diff in F.A.S. until gray matter from white; wash, diff in sodium borate-potassium ferricyanide; wash,ammonia H2O | MYELIN SHEATH = blue-blue/black; background = myelin | Gray matter and demyelinated white matter should be light brown and contrast sharply with myelinated white matter (opposite of what you'd normally see!)
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| LUXOL FAST BLUE | demonstrate myelin in tissue sections; as axon degenerates so does myelin gets broken down | Sulfonated copper phthalocyanine, alcohol soluble; lipoproteins stained via acid-base salt formation | 10%NBF; 10-15u | section of spinal cord/medulla | LUXOL Blue incubate over night; rinse in 95%ROH; Diff in Lithium Carbonate; continue diff in 70%ROH until gray/white; wash, finish diff brief in LiCarb,70ROH until greenish blue of white sharp; run through | MYELIN= blue; Background & demyelinated= colorless | *CAN COMBINE /CRESYL ECHT VIOLET to stain both myelin sheath and nissl substance
*can combine w/ holmes to stain myelin sheath and nerve fibers
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| HOLZER | demonstrate glial fibers and areas of gliosis | glial fibers w/ cyrstal violet and resistant to decolorization w/ alkaline aniline-chloroform | 10% NBF; paraffin at 6-8u | section of cerebral cortex (not spinal cord) for demonstration of glial fibers | fresh phosphomolybdic acid; cover sections w/ abs ROH-chloroform (translucent); while wet cover w/ crystal violet stain; 10% KBr; Dry; differentiated in aniline oil/chloroform; was in xylene, mount | GLIAL FIBERS = blue, bakground = very pale blue to colorless | carefule of aniline oil = SENSITIZER
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| CRESSYL ECHT VIOLET | identify neurons in tissue sections of demonstrate LOSS of Nissl substance(basophilic material in cytoplasm; loss in injured neuron) | Because of RNA in Nissl bodies, very basophilic and sharply stained w/ basic aniline dyes (pH & diff provides specificity of Nissl) | 10%NBF; 6-8u paraffin | spinal cord | Stain in CRESYL ECHT VIOLET; rinse, place in 95%ROH; Transer to AbsROH; Xylene, Balsam-Xylene; Diff in 100%ROH, microscop; repeat diff until background colorless | Nissl, Nuclei (basophilic) = Blue to Purple Background = Colorless | Diff repeat until background is colorless; change alcohol after balsam-xylene frequently; can add acetic acid to staining solution (pH2.5) and not have to use balsam-xylene
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| BODIAN | Nerve fibers in tissue | Protargol(Ag) impreg, Cu destain connective; hydroquinone reduce silver; toned in AuCl or Oxalic; Sodium thosulfate removes unreduced silver | 10%NBF; 6-8u paraffin | peripheral nerve or cerebral cortex (nerves should NOT be in cross section) | Protargol,copper incubate; rinse; reduce (hydroq);rinse, tone;develop in oxalic acid until gray bkgrd; rinse, treat w/ sodium thio; counter w/ aniline blue; run through | NERVE FIBERS/NUCLEI = black; Background = light gray or blue | NEED chemically glassware or you get a dirty background!)
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| HOLMES SILVER NITRATE | Nerve fibers and neurofibrils in tissue | Argyrophil silver method; alkli used when compared to Bodian | 10%NBF; 10-15u paraffin | Cerebral cortex | Same as Bodian, except first steps are silver nitrate in dark, then impreg solution | AXONS AND NERVE FIBERS; NEUROFIBRILS = black | *CAN COMBINED w/ LUXOL FAST BLUE to stain both myelin (blue/green) and axons/nerve fibers (black)
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| BIELCSCHOWSKY-PAS | demonstrate presense of neurfibrillary tangles and senile plaques in alzheimer's | use of ammoniacle Ag solution, deposit on neurofibrils and axons; reduced by formaldehyde in developer; toned w/ Gold chloride eliminating background; SCHIFF RXN stains basement membrane AND AMYLOID | 10%NBF; paraffin at 8-10u | tissue from CNS (w/ senile plaques and neurofibrillary tangles) | frese ammoniacal Ag; place slides in 20% Ag in dark; wash, ammoniacal silver at room temp; ammonia H2O and then more ammoniacal Ag; devop w/ formaldehyde solution; tone in AuCl until first gray; sodium thio to remove unreduced; PAS rxn; run through | NEURO FIBRILLARY TANGLES AND PERIPHERAL NEURITES OF NEURITIC PLAQUES = dark black; AXONS= black; AMYLOID (PLAQUE CORES AND VASCULAR) = magenta; LIPOFUSCIN = magenta | *MICROWAVE TECHNIQUE of Bielchowsky same, no PAS: Axons, Cytoplasmic neuro fibrils = brown to black; Neurofibril tangles and plques of Alzheimer's = dark brown or black; neuromelanin = black; lipofuscin = brown or black
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| SEVIER-MUNGER MODIFICATION OF BIELSCHOWSKY: Sevier-Munger Modification | modified Bielschowsky; demonstrate nerve fibers an dpresence of neurofibrillary tangles and senile plaques in alzheimers; can be used for demonstrating granules of some carcinoid tumors | tissue is impregnated with AMMONIACAL SILVER; reduced by formaldehyde; yellow background remains (no tone) | 10% NBF; paraffin at 6-8u | tissue from CNS | 1)Incubate in 20% Ag nitrate;2) formalin to ammoniacal silver solution and pour on slides develp untill golden brown; rinse, sodium thiosulfate to remove unreduced; run through | NERVE ENDINGS & NEUROFIBRILS = black; Neurofibrillary tangles and peripheral neurites of neuritic plaques = black | reliable; ARGYROPHIL stain
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Created by:
Miellee
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