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HT/HTL: Chap9

NERVE

SPECIAL STAINPURPOSEPRINCIPLEFIXATIVE/TECHQ.C.REAGENTS/PROCEDURERESULTSNOTES
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
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;
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!)
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
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
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
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!)
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)
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
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
Created by: Miellee