Nucleic Acids and Enzymes

Feulgen reaction purpose (2)

demonstrate DNA, DNA quantification for tumor pathology

Feulgen reaction Mode of Action

HCl causes hydrolysis of DNA, removes purine bases (A and G), creates aldehyde groups that can be stained with Schiff

Feulgen Reaction reagents

Hydrozyler:

Primary stain:

Differentiator:

Counter stain:

Hydrozyler: HCl

Primary stain: Schiff reagent

Differentiator: sulfurous acid

Counter stain: light green

Feulgen reaction results

reddish purple:

light green:

reddish purple: DNA

light green: cytoplasm

Fixative rules for Enzymes (2 exceptions)

No fixatives used except:

Naphthol ASD chloroacetate esterase uses methanol with formaldehyde

phosphorylase uses cold acetone

Fixative rules for nucleic acids (1 exception)

10% NBF used except:

Feulgen uses any fixative EXCEPT bouins

Feulgen reaction QC

thickness:

Feulgen reaction QC

thickness: 4 um

Methyl green pyronin y purpose (2)

• differentiate between RNA and DNA

• identify plasma cells or immunoblasts in tissue

methyl green pyronin y mode of action

DNA is more polymerized so methyl green binds to it, and RNA is less polymerized so pyronin y binds to it

methyl green pyronin y QC:

thickness:

section must contain:

thickness: paraffin, 4 um

section must contain: plasma cells

methyl green pyronin y reagents:

pH:

nucleic acid stain:

methyl green purifier:

pH: acetate buffer solution

nucleic acid stain: methyl green pyronin y staining solution

methyl green purifier: chloroform

methyl green pyronin y results:

green to blue green:

red to rose:

mint green:

pale pink to colorless:

intense red:

green to blue green: DNA and nuclei

red to rose: RNA

mint green: goblet cells

pale pink to colorless: backgrounds

intense red: immunoblast and cell cytoplasm

May grunwald giemsa purpose (2)

• diffferentiate cells in bloody tissue

• demonstrate microorganisms

may grunwald giemsa mode of action

combining basic methylene blue and acidic eosin gives a wide color range due to impurities

may grunwald giemsa QC:

thickness:

tissue:

thickness: paraffin, 3-4 um

tissue: spleen

may grunwald giemsa reagents:

primary stain:

secondary stain:

differentiator:

secondary fixative:

primary stain: giemsa solution

secondary stain: jenner solution

differentiator: acetic acid

secondary fixative:methanol

may grunwald giemsa results:

blue:

pink/gray/blue

blue: nuclei and microorganisms

pink/gray/blue: white blood cells

alpha naphthyl acetate esterase purpose (2)

• differentiate between type 2 muscle and neurogenic atrophy

• demonstrate motor end plates and lysosomes

alpha naphthyl acetate esterase mode of action (3)

• esterases hydrolyze aliphatic and aromatic ester bonds

• classified as specific or nonspecific

• stains histiocytes and acetylcholinesterase in denervated fibers

alpha naphthyl acetate esterase equation (3)

basic fuchsin + sodium nitrate → hexazotized pararosaniline

alpha naphthyl acetate + water —-esterases→ alpha naphthol + acetic acid

alpha naphthol + hexazotized pararosaniline → azo dye

alpha naphthyl acetate esterase reagents:

incubation solution:

adjust pH:

incubation solution:

• pH 7.2 phosphate buffer

• pararosaniline

adjust pH: HCl or NaOH

alpha naphthyl acetate esterase results:

brick red:

pale yellow:

dark red-brown:

brick red:motor endplates

pale yellow: normal muscle fibers

dark red-brown: denervated muscle fibers, macrophages, lysosomes

naphthol ASD chloroacetate esterase (Leder) purpose

identify granulocytes in the classification of leukemias or chloromas

naphthol ASD chloroacetate esterase (Leder) mode of action (2)

• esterases hydrolyze aliphatic and aromatic ester bonds

• specific esterase stain

naphthol ASD chloroacetate esterase (Leder) reagents:

incubation solution:

adjust pH:

incubation solution:

• pararosaniline

• sodium nitrate

adjust pH: HCl or Na barbitol

naphthol ASD chloroacetate esterase results:

bright red:

blue:

unstained:

bright red: positive cells, granulocytes, mast cells

blue: nuclei

unstained: backgrounds

atpase purpose (2)

• differentiate between type 1, 2A, 2B muscle fibers

• separate myopathic from nuropathic processes

atpase mode of action (2)

• ATPase is pH dependent, on the type of muscle fiber

• metal precipitation of hydrolyzed orthophosphoric acid with calcium

atpase equations (4)

ATP + water —-ATPase→ ADP + H3PO4 + energy

H3PO4 + CaCl2 → CaHPO4 + 2H + 2Cl

CaHPO4 + CoCl2 → CoHPO4 + CaCl2

CoHPO4 + (NH4)2S → CoS + (NH4)2HPO4

atpase reagents:

adjust pH:

incubation solution:

• activate muscle and inhibit mitochondrial atpase:

adjust pH: HCl or NaOH

incubation solution:

• sodium barbitol

• activate muscle and inhibit mitochondrial atpase: calcium chloride

atpase results:

pH 9.4 slide:

• dark:

• light:

pH 4.3 slide:

• dark:

• light:

pH 4.6 slide:

• dark:

• light:

• medium:

pH 9.4 slide:

• dark: type 1

• light: type 2

pH 4.3 slide:

• dark: type 1

• light: type 2

pH 4.6 slide:

• dark: type 1

• light: type 2A

• medium: type 2B

acid phosphatase purpose (2)

• demonstrate inflammatory cells/lysosomes in muscle biopsies

• demonstrate acid maltase deficiency: pompe disease, lysosomal storage disease

acid phosphatase mode of action

• acid phosphatase hydrolyzes esters of orthophosphoric acid

acid phosphatase equations (2)

naphthol ASBI phosphate + water —acid phosphatase→ naphthol ASBI + orthophosphatate

naphthol ASBI + hexazotized pararosaniline → red azo dye

acid phsophatase reagents:

incubation medium:

pH:

counterstain:

incubation medium:

• pararosaniline hydrochloride

• sodium nitrate

pH: veronal acetate buffer 5.0

counterstain: methyl green

acid phosphatase results:

red:

green:

red: sites of acid phosphatase activity

green: background

alkaline phosphatase purpose (2)

• detection of regenerating muscle fibers

• diagnose inflammatory myopathy

alkaline phosphatase mode of action

• alkaline phosphatase hydrolyzes esters of orthophosphoric acid

alkaline phosphatase equations (2)

naphthol ASBI phosphate + water —alkaline phosphatase→ naphthol ASBI + orthophosphatase

naphthol ASBI + fast red violet → red azo dye

alkaline phosphatase reagents:

incubation mediun:

• pH:

• primary stain:

counterstain:

incubation mediun:

• pH: tris buffer 8.74

• primary stain: fast red violet

counterstain: harris/mayer hematoxylin

alkaline phosphatase results

pink red to red:

blue:

pink red to red: sites of alkaline phosphatase activity

blue: nuclei

NADH diaphorase purpose (2)

• demonstrate abnormalities in mitochondria, z band material, sarcoplasmic reticulum

• demonstrate architectural changes in central cores, target fibers, nemaline rods, targetoid fibers

NADH disphorase mode of action (2)

• diahoresis reaction

• tetrazolium salts accept hydrogen, but is reduced by flavoprotein enzymes

NADH disphorase equations (2)

NADH —-disphorase→ NAD+ + H

NBT (nitro blue tatrazolium) + H → formazan

NADH diaphorase reagents

incubation medium:

• pH:

• primary stain:

incubation medium:

• pH: phosphate buffer 7.4

• primary stain: NBT (nitro blue tetrazolium)

NADH diaphorase results:

dark purple deposits:

dark purple:

light purple:

dark purple deposits: sites of NADH diaphorase enzyme activity

dark purple: type 1 muscle fibers

light purple: type 2 muscle fibers

QC Rules for enzymes: cut at ____ um and use _____ tissue (1 exception)

• ___ uses spleen as a control

QC Rules for enzymes: cut at 10 um and use skeletal muscle tissue

• naphthol chloroacetate esterase (Leder stain) uses spleen as a control

QC Rules for nucleic acids: cut at ____ um AND:

• ___ must contain plasma cells

• ___ uses spleen tissue

QC Rules for nucleic acids: cut at 4 um AND:

• methyl green pyronin y must contain plasma cells

• may grunwald giemsa uses spleen tissue

succinic dehydrogenase (SDH) purpose

• identify the source of NADH diaphorase activity, because only mitochondria show positive SDH activity

succinic dehydrogenase (SDH) mode of action

• SDH substrate is incubated with nitro blue tetrazolium and formazan pigment is created

succinic dehydrogenase (SDH) equations (2)

succinate —succinic dehydrogenase (SDH)→ fumarate + hydrogen

NBT (nitro blue tetrazolium) + hydrogen -→ formazan

succinic dehydrogenase (SDH) reagents:

incubation medium:

• pH:

rinse:

secondary fixative:

incubation medium:

• NBT solution

• pH: phosphate buffer 7.6

rinse: physiological saline

secondary fixative: formaline saline

succinic dehydrogenase (SDH) results:

blue:

blue: sites of succinic dehydrogenase (SDH) activity

cytochrom c oxidase (COX) purpose

identify cytochrome c oxidase activity

cytochrome c oxidase (COX) mode of action

diaminobenzidine (DAB) is an electron donor to show cytochrome c oxidase (COX) activity which will form a soluble polymer

cytochrome c oxidase (COX) reagents:

incubation medium:

• blocking:

• pH:

mounting media:

incubation medium:

• blocking: catalase

• pH: sorenson buffer 7.4

mounting media: toluene based

cytochrome c oxidase (COX) results

brown:

brown: sites of cytochrome c oxidase (COX) activity

phosphorylase purpose

• diagnose mcardle disease

phosphoylase mode of action

• phosphorylase creates unbranched amylose, whose length is proportional to phosphorylase activity

phosphorylase reagents:

incubation medium:

• prevent excessive branching:

• primer:

• pH:

• activator:

visualizer:

mounting medium:

incubation medium:

• prevent excessive branching: alcohol

• primer: glycogen

• pH: acetate buffer 5.9

• activator: insulin

visualizer: gram iodine

mounting medium: iodine-glycerin medium

phosphorylase results:

blue/puple:

colorless:

blue/puple: phosphorylase activity

colorless: mcardle disease

modified gomori trochrome reagents:

gomori trichrome solution:

• myofibril stain:

• muscle feature stain:

• differentiator for muscle:

• pH:

differentiator at end:

nuclei stain:

gomori trichrome solution:

• myofibril stain: fast green fcf

• muscle feature stain: chromotrope 2R

• differentiator for muscle: phosphotungstic acid

• pH: glacial acetic acid

differentiator at end: acetic acid

nuclei stain: harris hematoxylin

modified gomori trichrome results:

red/purple:

blue/green:

red:

red/purple: nuclei

blue/green: myofibrils

red: intermyofibrillar sarcoplasm, myelinated nerve twigs, nemaline rods, abnormal mitochondria

modified Gomori trichrome purpose

identify structural abnormalities in muscle to check for mitochondrial dysfunction

modified gomori trichrome mode of action

• large tungstate ion is taken up by thick collagen fibers and it stains this complex

• small chromotrope 2R molecules taken up by thin muscle fibers, and it stains the complex