Chapter 9: Nerve Flashcards

Question

Bielschowsky-PAS Purpose

Answer 1

To demonstrate nerve fibers, neurofibrillary tangles, and senile plaques in Alzheimer disease

Answer 2

1. impregnate with ammoniacal silver which deposits on neurofibrils and axons 2. silver is reduced to metallic silver by formaldehyde in the developer 3. Gold chloride tones the tissue and removes yellow 4. sodium thiosulfate removes unreduced silver background 5. schiff reaction (PAS) stains basement membranes and amyloid in the plaques

Answer 3

10% NBF | Control: CNS tissue containing plaques and tangles

Answer 4

1. Prepare ammoniacal silver solution 2. Deparaffinize, hydrate 3. Place in silver nitrate solution in the dark at room temp 4. Wash with distilled water 5. Place in ammoniacal silver solution 6. Wash in ammonia water 7. While slides are in ammonia water, add 2 drops of developer to the ammoniacal silver solution from step 5 8. place slides in ammoniacal/developer solution. The tissue should turn brown in about 3 minutes 9. Wash well in ammonia water, then distilled water 10. Tone in gold chloride until grey appears 11. Wash in ammonia water, then rinse in distilled water 12. Place in sodium thiosulfate 13. Wash in running tap water 14. Rinse well in distilled water 15. Place sections in 1% periodic acid 16. Rinse in distilled water 17. Place in schiff reagent 18. Wash in tap water 19. Dehydrate, clear, coverslip

Answer 5

Neurofibrillary tangles: dark black Peripheral neurites and plaques: dark black Axons: black Amyloid (plaque cores and vasculature): magenta Lipofuchsin: magenta

Answer 6

Demonstrate nerve fibers, tangles, and plaques in Alzheimer's

Answer 7

Sections are not toned with gold chloride so yellow background remains

Answer 8

10% NBF | Control: CNS with plaques and tangles

Answer 9

1. Deparaffinize, hydrate 2. Place slides in 1% Silver Nitrate, dip several times and incubate in warm solution for 15 minutes 3. Place slides in distilled water 4. 1% silver nitrate from step 2 + 28% ammonium drops until precipitate clears, then 5% silver nitrate drops until solution becomes slightly cloudy 5. microwave slides in the solution, dip slides and incubate in warm solution 6. Place slides in 1% ammonium hydroxide 7. add 3 drops developer to ammoniacal silver from step 5, place slides in solution until tissue turns brown 8. Place slides in 1% ammonium hydroxide 9. Rinse in distilled water 10. Wipe silver mirror off both sides of slide without disturbing the tissue 11. place slides in 2% sodium thiosulfate 12. Rinse slides in distilled water 13. Dehydrate, clear, coverslip

Answer 10

``` Axons: brown to black Cytoplasmic neurofibrils: brown to black Tangles and plaques: dark brown or black Neuromelanin: black Lipofuchsin: brown or black ```

Answer 11

Use chemically cleaned glassware, non-metallic forceps Uses much less silver nitrate than traditional Bielschowsky and stains tangles and plaques better

Answer 12

Demonstrate nerve fibers, tangles, and plaques for Alzheimer's

Answer 13

Ammoniacal silver impregnation Reduced by formaldehyde developer No toning with gold chloride so yellow background remains Sodium Thiosulfate removes unreduced silver

Answer 14

10% NBF Control: CNS with tangles and plaques 6-8uM sections

Answer 15

1. Deparaffinize, hydrate 2. Preheat 20% silver nitrate, then add slides to warm silver solution and incubate in the oven 3. Rinse one slide at a time in distilled water and place in a clean, dry staining jar 4. add 10 drops formalin, while agitating, to working ammoniacal silver solution, pour over slides and develop until golden brown. Check microscopically, do not wash while checking, keep in motion while developing to avoid precipitation 5. Rinse slides well in tap water 6. Place in sodium thiosulfate 7. Wash well in tap water 8. Dehydrate, clear, coverslip

Answer 16

Nerve endings and neurofibrils: black | Tangles, peripheral neurites, and plaques: black

Answer 17

Very reliable and reproduce-able technique Concentration of ammonium hydroxide and formalin are critical leave a few particles of silver, do not add extra ammonia This is an argyrophil stain that is also useful fro demonstrating the granules of some carcinoid tumor cells

Answer 18

Demonstrates presence of tangles, plaques, threads in addition to parenchymal and amyloid deposition in Alzheimer's disease

Answer 19

Fluorescent visualization of amyloid Pretreatment with potassium permanganate, and bleaching with potassium metabisulfate and oxalic acid Treatment with sodium hydroxide and hydrogen peroxide, which remove lipid autofluorescence for better definition of pathological lesions Visualization is better than routine Thioflavin S ans is not affected by prolonged fixation More sensitive than silver methods Faster and cheaper than silver

Answer 20

10-20% NBF Control: CNS with plaques and tangles 6um sections air dried overnight, then 10 minutes in 60C oven

Answer 21

1. Deparaffinize, hydrate 2. Rinse and old in distilled water 3. cover tissue with 0.25% potassium permanganate 4. wash slides in running tap water 5. treat slides with 1% potassium bisulfate 6. wash in running tap water 7. place in sodium hydroxide-peroxide 8. wash in running tap water and then filtered water 9. place slides in 0.25% acetic acid 10. wash slides in running tap water 11. place slides in 50% alcohol 12. place in Thioflavin S 13. rinse slides in 50% alcohol with agitation 14. Rinse in 95% alcohol 15. completely dehydrate in absolute alcohol and clear in xylene. Mount with non-fluorescent mounting medium 16. View slides on a fluorescent filter set that incorporates a blue-violet excitation filter

Answer 22

tangles, neurites, threads, plaque amyloid, and cerebrovascular amyloid: bright green Diffuse plaques and extracellular plaques: paler yellow green PSP tangles and Pick bodies: not well demonstrated

Answer 23

Float tissue on pre-heated DI water bath Mount sections with cytoseal 60 Staining is stable for several months at room temp

Answer 24

Demonstration of glial fibers (also used to demonstrate muscle striations)

Answer 25

High ratio of PTA to hematein 20:1 so that tungsten binds all available hematein to form a blue lake which stains glial fibers, nuclei, and myelin. Red brown or salmon color of neurons is due to the PTA alone. Overuse of alcohol will fade the red-brown

Answer 26

10% NBF 6-8uM sections Control: Cerebral cortex (not spinal cord)

Answer 27

1. Deparaffinize, hydrate 2. Mordant the sections overnight at room temp in Zenker solution containing acetic acid 3. Wash sections in running water 4. Place in Lugol Iodine. Do not use sodium thiosulfate because it may impair subsequent staining 5. Decolorize the sections in 95% alcohol for 1 hour 6. Rinse rapidly in distilled water 7. Place in 1% potassium permanganate 8. Wash in running tap water 9. Decolorize the sections in 5% oxalic acid 10. Wash in running tap water 11. Stain in PTAH overnight 12. Dehydrate, clear, coverslip

Answer 28

Glial fibers: blue Nuclei: blue Neurons: salmon Myelin: blue

Answer 29

Replaced by immunohistochemical methods Don't over rinse in EtOH Don't use sodium thiosulfate or stain won't work

Answer 30

To demonstrate glial fibers and areas of gliosis (glial damage)

Answer 31

Glial fibers are stained with crystal violet and are resistant to decolorization with an alkaline aniline-chloroform mixture

Answer 32

10% NBF 6-8uM sections Control: Cerebral cortex, not spinal cord

Answer 33

1. Deparaffinize, hydrate 2. place in PMA-alcohol 3. Drain off excess fluid, place slides in a staining rack and cover the sections with absolute alcohol-chloroform mixture. The tissue should become translucent 4. While sections are still wet, cover them with the crystal violet stain 5. Replace the stain with 10% potassium bromide 6. Blot the sections dry and then allow them to air dry 7. Differentiate in aniline-oil+chloroform+ammonium hydroxide differentiating solution 8. Wash in several changes of xylene. Repeat steps 7 and 8 until the background is very pale or colorless 9. Mount with synthetic resin

Answer 34

Glial fibers: blue | Background: very pale blue to colorless

Answer 35

Crystal Violet precipitate may be removed with straight aniline oil Aniline oil and chloroform are both very hazardous; use in a chemical fume hood The aniline oil-chloroform-ammonium hydroxide differentiating solution resists decolorization

Answer 36

Demonstrate astrocytes

Answer 37

Astrocytes are selectively stained with the Cajal gold sublimate method on frozen sections

Answer 38

Formalin ammonium bromide for 2-25 days, rinse and place in formalin ammonium bromide for 2 days if previously fixed with 10% NBF 20-30uM Sections, free floating, no slides Control: cerebral cortex, not spinal cord

Answer 39

1. wash free floating frozen sections in several changes of distilled water 2. Transfer the sections to the gold sublimate solution (gold chloride + mercuric chloride) and incubate in the dark for 4 hours, sections should turn purple 3. Wash well in distilled water 4. Treat with 5% sodium thiosulfate 5. Wash in distilled water 6. Carefully mount the sections on slides, blot and dehydrate 7. Clear in xylene and mount with synthetic resin

Answer 40

Astrocytes: black

Answer 41

Tissue sections better if washed in tap water for 30 minutes before freezing chemicals must be pure; brown chloride is preferred over yellow gold chloride Protoplasmic (grey matter) astrocytes lose stainability after prolonged fixation Staining solution should not exceed 30C mixture of mercuric chloride and gold chloride is essential for astrocyte demonstration mercuric chloride is extremely toxic and an environmental hazard

Answer 42

Demonstration of myelin Myelin degeneration, such as in syphilis

Answer 43

Mordant-hematoxylin solutions attaches to the phospholipid component of the myelin sheath, which has an affinity for the cationic (+) lake Regressive technique 1. Ferric ammonium sulfate: excess mordant differentiation removes most of the excess dye 2. Borax ferricyanide: oxidizer differentiation which removes any remaining non-specifically bound hematoxylin lake and forms a colorless oxidation product. Only myelin sheath and red blood cells remain stained

Answer 44

10% NBF 10-15uM Sections Control: Spinal cord or medulla

Answer 45

1. Deparaffinize, hydrate 2. Transfer sections to staining solution for 30 minutes at 54-56C 3. Wash in tap water 4. Differentiate in 4% ferric ammonium sulfate until the gray matter can just be distinguished from the white matter and the stain is removed from the slides 5. Wash in tap water 6. Complete differentiation in the sodium borate-potassium ferricyanide solution. This should be checked until the grey and white matter are sharply defined 7. Wash in tap water 8. Treat sections in diluted ammonia water 9. Wash in distilled water 10. Dehydrate, clear, coverslip

Answer 46

Myelin sheath: blue to blue-black | Background: light tan

Answer 47

Gray matter and demyelinated white matter should appear light brown and contrast sharply with blue-black myelinated white matter Easy macroscopic differentiation Naturally ripen for 6 months or chemically ripen with ferric ammonium sulfate

Answer 48

Demonstrate myelin

Answer 49

Similar to alcian blue, but is alcohol rather than water soluble. Staining is accomplished through an acid-base reaction in which the base of the myelin lipoprotein swaps with the base of the dye

Answer 50

10% NBF 10-15uM Sections Control: spinal cord or medulla

Answer 51

1. Deparaffinize, hydrate 2. Place slides in LFB (Luxol Fast Blue) and incubate overnight at 56-58C 3. Rinse in 95% alcohol to remove excess stain (LFB is alcohol soluble) 4. Rinse in water 5. Begin differentiating by immersing slides in lithium carbonate solution 6. Continue differentiation in 70% alcohol until gray matter and white matter can be distinguished. Do not over differentiate 7. Wash in distilled water 8. Finish differentiation by rinsing briefly in lithium carbonate and then through several changes of 70% alcohol until the greenish blue of the white matter contrasts sharply with colorless grey matter 9. Rinse in distilled water 10. Dehydrate, clear, coverslip

Answer 52

Myelin: blue to blue-green Background: colorless

Answer 53

Gray matter and demylinated white matter should be almost colorless and contrast sharply with blue stained myelinated white matter Quality of the stain (contrast) can be determined macroscopically Differentiate until gray matter is almost colorless

Answer 54

Demonstrate myellin and nerve fibers in the same section

Answer 55

LFB staining is accomplished through an acid-base reaction in which the base of the myelin lipoprotein swaps with the base of the dye Holmes is an argyrophil silver method that requires chemical reduction Gold chloride toner, Oxalic acid reducer, and sodium thiosulfate

Answer 56

10% NBF 10-15uM Sections Control: Cerebral cortex or longitudinal section of peripheral nerve

Answer 57

1. Deparaffinize, hydrate 2. 20% silver nitrate in the dark at room temp 3. make impregnating solution (boric acid+borax+silver nitrate+pyradine) 4. Take slides out of 20% silver nitrate and wash in distilled water 5. 37C overnight incubation in impregnating solution 6. shake off excess fluid, and place in reducer (hydroquinone+sodium sulfite) 7. Wash in running water, then distilled water 8. Tone in gold chloride. Solution may be reused until a brown precipitate forms or the solution becomes cloudy 9. Rinse in distilled water 10. Place in oxalic acid, stop when sections are blue-black 11. Rinse in distilled water 12. Place slides in sodium thiosulfate 13. Wash in tap water 14. place in 95% alcohol 15. Stain in LFB overnight at 60C 16. place in 95% alcohol 17. Place in distilled water 18. Place in lithium carbonate 19. Differentiate in 70% alcohol 20. Rinse in distilled water (repeat steps 18-20 until desired differentiation is achieved) 21. Dehydrate, clear, coverslip

Answer 58

Myelin: blue to green | Axons and nerve fibers: black

Answer 59

Demonstrate both myelin and Nissl substance

Answer 60

LFB staining is accomplished through an acid-base reaction in which the base of the myelin lipoprotein swaps with the base of the dye Cresyl echt violet uses a basic aniline dye to stain RNA blue, thus demonstrating the RNA in the rough endoplasmic reticulum which comprises the Nissl substance

Answer 61

10% NBF 10-15uM sections Control: spinal cord or medulla

Answer 62

1. Deparaffinize, hydrate 2. Place slides in LFB, incubate overnight in 56-58C 3. rinse in 95% alcohol to remove excess stain 4. Rinse in distilled water 5. Begin differentiation by immersion in lithium carbonate 6. Continue differentiation in 70% alcohol until grey and white matter can be distinguished 7. Rinse in distilled water 8. Finish differentiation by rinsing briefly in lithium carbonate and then through several changes of 70% alcohol until sharp contrast is achieved 9. Rinse in distilled water 10. Add acetic acid, filter, and preheat cresyl echt violet. Place slides in cresyl echt violet for 6 minutes, keep warm while staining. 11. Differentiate in several changes of 95% alcohol 12. Dehydrate, clear, coverslip

Answer 63

Myelin: blue (from LFB) Nissl substance: violet (from cresyl) Nuclei: violet (from cresyl)

Answer 64

If you don't add acetic acid to the CEV, you will get a diffuse violet background stain Staining of Nissl will be reduced if you don't heat the CEV prior to staining CEV counterstain intensifies the myelin sheath stain

Answer 65

Demonstrate myelin sheath, basement membranes, senile plaques, fungi, and corpora amylacea The various stains sharpen and complement each other

Answer 66

LFB staining is accomplished through an acid-base reaction in which the base of the myelin lipoprotein swaps with the base of the dye Periodic acid oxidizes reactive groups to form aldehydes (-CHO) Basic fuschin + sulfurous acid = leucofuschin/Schiff's reagent which binds to exposed aldehyde groups Water washes away the sulfur resulting in the rose chromophore

Answer 67

10% NBF 10-15uM Sections Control: Cerebral cortex or medulla

Answer 68

1. Deparaffinize, hydrate 2. Incubate in LFB overnight at 56-58C 3. Rinse in 95% alcohol to remove excess stain 4. Rinse in distilled water 5. Begin differentiation by placing in lithium carbonate 6. Continue differentiation in 70% alcohol until grey and white matter become distinguished 7. Wash in distilled water 8. Finish differentiation by rinsing briefly in lithium carbonate and then through several changes of 70% alcohol until sharp contrast is achieved (repeat 7-8 as necessary) 9. Rinse in distilled water 10. Place in periodic acid (aldehyde formation) 11. Place in distilled water 12. Place in schiff solution 13. Wash in tap water 14. Stain with Harris hematoxylin 15. Wash in tap water (if background is not clear dip once in acid-alcohol and wash, if nuclei are not dark blue to purple dip briefly in dilute ammonium hydroxide and wash) 16. Dehydrate, clear, coverslip

Answer 69

``` Capillary basement membranes: Rose Fungi: Rose Corpora amylacea: Rose Senile plaques: Rose Myelin Sheath: Blue to blue-green Nuclei: purple ```

Answer 70

CNS: brain and spinal cord PNS: everything else

Answer 71

Somatic: voluntary Autonomic: involuntary

Answer 72

Neuronal cell bodies and processes Glial cells and processes Myelin sheath

Answer 73

Cellular unit of the nervous system, conduct information vial electrochemical impulses

Answer 74

``` Cell body containing nucleus Dendrites Axon Myelin sheath Node of Ranvier ```

Answer 75

Basophilic: stains blue/black with hematoxylin, or basic aniline dyes like thionin and cresyl echt violet Large aggregates of rough endoplasmic reticulum with RNA content being stained

Answer 76

"Nerve glue" like a connective tissue for the CNS Insulate neurons except at synapses produce the myelin sheath

Answer 77

Oligodendroglia/oligodendrocytes Astrocytes Microglia Ependymal Cells

Answer 78

Produce and maintain myelin sheath in the CNS

Answer 79

Protoplasmic(gray) and fibrous(white) help in scar formation after brain injury support nerve fibers Star shaped (stellate)

Answer 80

fixed phagocytic cells found throughout the brain and spinal cord (immune response)

Answer 81

Epithelium with a brush border that acts as the blood/brain barrier

Answer 82

Fatty insulation around neurons Stains with lipid stains Deposited by oligodendrocytes on multiple neurons in the CNS Deposited by Schwann cells on individual neurons in the PNS Typically demonstrated with Luxol fast blue and iron hematoxylin