Routine Staining (textbook)

Ultrastructure of the Cell

• 1665: ___

• 1838: ___

• 1839: ___

• Eukaryotic cells divided into:

  • ___

  • ___

Ultrastructure of the Cell

• 1665: Robert Hook finds cells in cork

• 1838: Matthias Schleiden finds cell is basic unit of vegetables

• 1839: Theodore Schwann finds cell is basic unit of life

• Eukaryotic cells divided into:

  • Cytoplasm

  • Nucleus

Ultrastructure of the Cell

The Nucleus

• Nucleus looks different whether it is going thru ___ or ____

  • Gap 0: ___

  • Gap 1: ___

  • S: ___

  • Gap 2: ___

• Nuclei are ___and look ___

Ultrastructure of the Cell

The Nucleus

• Nucleus looks different whether it is going thru mitosis or not (interphase):

  • Gap 0: cell stops dividing

  • Gap 1: cell grows, synthesize RNA and proteins

  • S: DNA duplicates

  • Gap 2: Fix mistakes from S

• Nuclei are basophilic and look purple with an H&E stain

Ultrastructure of the Cell

The Nucleus

• Nuclear Membrane

  • Made up of ___, separated by ___

  • ___ lines the ___, except at ____

  • ___ stains the ___

Ultrastructure of the Cell

The Nucleus

• Nuclear Membrane

  • Made up of two membranes, separated by perinuclear space

  • Heterochromatin lines the inside membrane, except at nuclear pores

  • Hematoxylin stains the heterochromatin

Ultrastructure of the Cell

The Nucleus

• Nuclear Pores

  • ____ in the ___

  • ___ and ___ move out of nucleus

  • ___ and ___ move into nucleus

Ultrastructure of the Cell

The Nucleus

• Nuclear Pores

  • Openings in the nuclear membrane

  • RNA and proteins move out of nucleus

  • Carbs and lipids move into nucleus

Ultrastructure of the Cell

The Nucleus

• Nucleolus

  • ___philic overall

  • 80-90% protein (___philic), will stain w ____ sometimes

  • Produces most of the ___ in the cell

Ultrastructure of the Cell

The Nucleus

• Nucleolus

  • Basophilic

  • 80-90% protein (acidophilic), will stain w eosin sometimes

  • Produces most of the RNA in the cell

Ultrastructure of the Cell

The Nucleus

• Chromatin

  • ___chromatin is the ___ portion of chromosomes, and it is ___

    • Genetically ___

  • ___chromatin is the ___ portion of chromosomes, and it is ___

  • Cells will differ in stainability depending on amount of ____chromatin in the nucleus

    • ___ nuclei: intense staining

    • ___ nuclei: slight staining

    • ___ nuclei: scattered staining

  • Staining MIGHT be affected by ___

Ultrastructure of the Cell

The Nucleus

• Chromatin

  • Euchromatin is the extended portion of chromosomes, and it is unstainable

    • Genetically active

  • Heterochromatin is the condensed portion of chromosomes, and it is stainable

  • Cells will differ in stainability depending on amount of heterochromatin in the nucleus

    • Lymphocyte nuclei: intense staining

    • Neuron nuclei: slight staining

    • Epithelial nuclei: scattered staining

  • Staining MIGHT be affected by histones

Ultrastructure of the Cell

The Cytoplasm

• Most of the ___ of the cell happens here

Ultrastructure of the Cell

The Cytoplasm

• Most of the work of the cell happens here

Ultrastructure of the Cell

Plasmalemma

• ___ membrane/___ membrane

• Made of ___

• Surrounds ___ and ___

• Glycocalyx: ___

• Not seen in ___ but important to ___ reactions

Ultrastructure of the Cell

Plasmalemma

• Plasma membrane/trimellar membrane

• Made of phospholipids

• Surrounds cell and organelles

• Glycocalyx: glycoprotein outer surface of membrane

• Not seen in H&E but important to antigen-antibody reactions

Ultrastructure of the Cell

Mitochondria

• Not seen in ___

• Site of ___ for ___

• More mitochondria = more ___

Ultrastructure of the Cell

Mitochondria

• Not seen in H&E

• Site of oxidative reactions for cell energy

• More mitochondria = more energy needed

Ultrastructure of the Cell

Ribosomes

• ___ synthesis

• Location:

  • On ___

  • ___ in cytoplasm

  • ___ in cytoplasm

• Causes ___ tint in ___

  • looks localized due to ___

  • looks diffuse due to___

Ultrastructure of the Cell

Ribosomes

• Protein synthesis

• Location:

  • On rough ER

  • Single in cytoplasm

  • Clustered in cytoplasm

• Causes basophilic blue tint in cytoplasm

  • Localized due to rough ER

  • Diffuse due to free ribsomes

Ultrastructure of the Cell

Endoplasmic Reticulum

• Used for ___

• ___ or ___ ER

• ___ Causes ___tint in ___

  • ___cells

  • ___cells

• Smooth ER does ___

Ultrastructure of the Cell

Endoplasmic Reticulum

• Used for transport of substances out of or around cell

• Granular/Rough or Agranular/smooth ER

• Rough ER Causes basophilic blue tint in cytoplasm

  • Plasma cells

  • Acinar cells

• Smooth ER does lipid synthesis

Ultrastructure of the Cell

Golgi Apparatus

• ___ in the cell

• Does not stain with ___

• Looks like a ___ around ___ in ___ cells

Ultrastructure of the Cell

Golgi Apparatus

• Packaging for transport in the cell

• Does not stain with H&E

• Looks like a halo around nucleus in plasma cells

Ultrastructure of the Cell

Centriole

• Does not stain with ___

• Creates ___ for cell ___

Ultrastructure of the Cell

Centriole

• Does not stain with H&E

• Creates spindles for cell division

Ultrastructure of the Cell

Lysosomes

• ___, ___, and ___

• Indigestible stuff is put in ___ "___"

  • Lipofuscin: ___

• Contains ___, ___,___

  • Endogenous: ___

  • Exogenous: ___

Ultrastructure of the Cell

Lysosomes

• Digestion, protection, and trash

• Indigestible stuff is put in vacuoles "residual bodies"

  • Lipofuscin: accumulation of residual bodies that shows up as yellow/brown pigment

• Contains vesicles, pigments, food

  • Endogenous: melanin, hemosiderin, lipofuscin, bilirubin

  • Exogenous: carbon, asbestos, tattoo ink, dust, metals

Staining Mechanisms

• Most staining reactions are both ___ and ___

• Adsorption: ___

  • Ionic/___: ___

  • Covalent: ___

    • how ___ bond

  • Coordinate: ___

    • how ___ bond

  • Hydrogen bond: ___

  • LDF

Staining Mechanisms

• Most staining reactions are both physical and chemical

• Adsorption: attraction of particles from solution to to surface of tissue

  • Ionic/salt linkage: dye and substance have different charges

  • Covalent: both atoms donate and share electrons

    • mordants

  • Coordinate: one atom donates and share electrons

    • Mordants

  • Hydrogen bond: H and N,O,F atoms

  • LDF

Staining Mechanisms

Nuclear Staining

• Two main mechanisms:

  • Staining with ___

  • Staining with ___

    • ___ are altered or removed

    • ___ is taken off and leaves ___ soluble

    • Metal mordants cannot be called ___ because they lack ___

      • ___

      • ___

Staining Mechanisms

Nuclear Staining

• Two main mechanisms:

  • Staining with basic/positively charges dyes

  • Staining with dyes followed by metal mordants

    • Nucleic acids are altered or removed

    • Protein on phosphate group is taken off and leaves DNA soluble

    • Metal mordants cannot be called basophilic because they lack acidic groups

      • Myelin

      • Mucopolysaccharides

Staining Mechanisms

Cytoplasmic Staining

Due to ___ portions of ___

• ___ dependent and peptides can be ___ or ___

  • Basic ___ amino group

  • Acidic ___ amino group

• Amphoteric

• How the peptide behaves is based on its ___

• IEP/isoelectric point: ___

  • Below pH 6: ___ protein, ___ dye attraction

  • Above pH 6: ___protein, ___ dye attraction

• Basophilic: ___

• Acidophilic: ___

• Eosin is (-)  and stains cytoplasm except when

  • ___: all of the tissue is stained

  • ___: will not stain (due to IEP changing)

Staining Mechanisms

Cytoplasmic Staining

• Due to charged portions of peptides

  • pH dependent and peptides can be (+) or (-)

    • Basic (+) COOH amino group

    • Acidic (-) NH2 amino group

  • Amphoteric

• How the peptide behaves is based on its net charge

• IEP/isoelectric point: when the net charge is neutral, pH of 6

  • Below pH 6: (+) protein, (-) anionic, acidic dye attraction

  • Above pH 6: (-) protein, (+) cationic, basic dye attraction

• Basophilic: substances that attract basic (+ charged) dyes

• Acidophilic: substances that attract acidic (- charged) dyes

• Eosin is (-)  and stains cytoplasm except when

  • pH is low: all of the tissue is stained

  • pH above 6: will not stain (due to IEP changing)

Staining Mechanisms

The Dyes

• Mosty derived from ___, ___, or ___

• Chromophore: ___

  • Double bonded: ___, ___, ___, ___, ___, ___, and ___

  • More of them, more ___

  • Easily ___, unsatisfied affinity for ___

• Chromogen: ___

• Auxochrome: ___

  • ___, ___, ___ and ___

  • Sulfonic acid used for ___ OR ___

  • Basic dye auxochrome is ___

  • Acidic dye auxochrome is ___or ___

• Basic (+) dyes are usually ___ salts

  • ___ and ___

• Acidic (-) dyes are usually ___ salts

  • ___and ___

• Amphoteric: ___

• Lysochromes: ___

• Physical staining: ___

• Natural dyes in Histotecnology: ___, ___, ___, ___

Staining Mechanisms

The Dyes

• Mosty derived from coal, tar, or benzene

• Chromophore: group of atoms in a molecule that give it color

  • Double bonded: CC, CO, CS, CN, NN, NO, and NO2

  • More of them, more pronounced the color

  • Easily reduced, unsatisfied affinity for H

• Chromogen: compound (usually benzene) containing a chromophore

• Auxochrome: ionizing group that allows dye to bind

  • Amino group, Sulfonic acid (weak), Carboxyl and hydroxyl groups

  • Sulfonic acid used for water solubility OR (+) -> (-)

  • Basic dye auxochrome is NH2

  • Acidic dye auxochrome is COOH or OH

• Basic (+) dyes are usually chloride salts

  • Crystal violet and safranin

• Acidic (-) dyes are usually sodium salts

  • Orange G and picric acid

• Amphoteric: functions as acidic or basic dye depending on pH and IPE

• Lysochromes: non-ionic dyes that dye by dissolving into the tissue

• Physical staining: transferring from the solvent solution to the tissue

• Natural dyes in Histotecnology: hematoxylin, carmine, orcein, saffron

Factors Affecting Dye Binding

• ___determines charge of tissue and dye, which impacts staining

• Increase in ___ = increase rate of stain

• Increase in ___ = increased dye binding

• ___ can increase OR decrease staining intensity

• Some ___ reduce stainability of tissue

  • ___ tissue increase basophilia

  • ___ tissue increase acidophilia

  • ___tissue is an intermediate

Factors Affecting Dye Binding

• pH determines charge of tissue and dye, which impacts staining

• Increase in temp = increase rate of stain

• Increase in concentration = increased dye binding

• Dissolved salts can increase OR decrease staining intensity

• Some fixatives reduce stainability of tissue

  • Formalin fixed tissue increase basophilia

  • Picric acid fixed tissue increase acidophilia

  • Ethyl alcohol fixed tissue is an intermediate

Factors Affecting Dye Binding

Differentiation

• Progressive staining: ___

• Regressive staining: ___

• Differentiated: ___

  • Basic dyes are differentiated by ___

  • Acidic dyes are differentiated by ___

  • Excess mordant is rectified by ___

  • Oxidizers: ___, ___

  • Solvents: ___ or ___

  • Mordant: ___

Factors Affecting Dye Binding

Differentiation

• Progressive staining: color intensity increases until desired outcome is reached

• Regressive staining: color intensity decreases until desired outcome in reached

• Differentiated: decolorized

  • Basic dyes are differentiated by weak acidic solutions

  • Acidic dyes are differentiated by weak alkaline solutions

  • Excess mordant: Verhoeff-Van Gieson procedure

  • Oxidizers: Potassium permanganate, chromic acid

  • Solvents: Water or alcohol

• Mordant: substances that act as a link between tissue and dye

The Nuclear Dyes

• Hematein: ___

• Hematin: ___

  • Via ___

• Hematein is formed by ___

  • Aka ___

  • Natural oxidation: ___, ___

  • Chemical oxidation: ___, ___, ___

    • ___, ___, ___

• Hematein is only a strong dye with a ___

• Hematein-___ complex is actually ___, despite staining ___

  • Sheehan: ___

  • Horobin: ___

  • Kiernan: ___

• The mordant should never ___

  • ___ are unstable

• ___/___ are most common mordants

• Lake: ___

• Non-oxidizing mordant: ___, ___

• Hemalum: ___

• Alum: ___

• ___ or ___ can differentiate

  • ___ rectifies overoxidation

  • ___ makes artifact

• Components of alum-hematoxylin solution

  • ___ for hematoxylin -> hematein

    • ___

  • ___ for pH adjusting and selectivity

  • ___ to prevent further oxidation

  • ___ to prevent evaporation

The Nuclear Dyes

• Hematein: oxidized dye of the campeachy/log wood

• Hematin: formalin pigment

  • Via breakdown of hemoglobin

• Hematein is formed by oxidizing hematoxylin

  • Aka ripening

  • Natural oxidation: Delafield, Ehrlich

  • Chemical oxidation: Harris, Mayer, Gill

    • Potassium permanganate, sodium iodate, mercuric oxide

• Hematein is only a strong dye with a metallic mordant

• Hematein-metal complex is actually anionic, despite staining anionic nuclei

  • Sheehan: binds to (+) histones OR (+) phosphate groups

  • Horobin: binds via nonionic forces

  • Kiernan: hemalum IS cation and stains DNA directly via phosphate anions

• The mordant should never oxidize the dye

  • Iron hematoxylins are unstable

• Ammonium/potassium aluminum sulfate are most common mordants

• Lake: mordant-dye solution

• Non-oxidizing mordant: phosphotungstic acid, phosphomolybdic acid

• Hemalum: hematein and aluminum sulfate mordant solution

• Alum: double metal sulfate mordant, usually with potassium or iron

• Acids or Aluminum can differentiate

  • Excess Aluminum rectifies overoxidation

  • Too much aluminum makes artifact

• Components of alum-hematoxylin solution

  • Oxidizing agents for hematoxylin -> hematein

    • Sodium iodate

  • Acids for pH adjusting and selectivity

  • Stabilizers to prevent further oxidation

  • Solvents to prevent evaporation

The Nuclear Dyes

• Harris hematoxylin

  • Hematoxylin

    • ---

      preservative

  • ___

    • ___ is mordant

  • ____

    • solvent

  • ___

    • Oxidizer

  • Used via___ OR ___

    • Use___ for acid

    • ____ hematoxylin is consistent

The Nuclear Dyes

• Harris hematoxylin

  • Hematoxylin

  • Absolute ethanol

    • preservative

  • Ammonium aluminum sulfate

    • Aluminum is mordant

  • DI water

    • solvent

  • Sodium iodate

    • Oxidizer

  • Used via acidified (selective staining) OR progressive

    • Use 1% HCl for acid

    • Acidified aluminum hematoxylin is consistent

The Nuclear Dyes

• Delafield Hematoxylin

  • ___

    • Aluminum is___

  • ___

  • Hematoxylin

    • ___

  • ___

    • preservative

  • ___

    • Prevents ___ and evaporation

  • ___

    • Preservative

  • Used ___

  • Testing to see if its good:

    • On paper:

      • OK: ___

      • Underoxidized: ___

    • In water:

      • OK: ___

      • Underoxidized: ___

The Nuclear Dyes

• Delafield Hematoxylin

  • Ammonium Aluminum sulfate

    • Aluminum is mordant

  • DI water

  • Hematoxylin

    • Physical oxidation

  • 95% alcohol

    • preservative

  • Glycerol

    • Prevents overoxidation and evaporation

  • 95% ethanol

    • Preservative

  • Used regressively

  • Testing to see if its good:

    • On paper:

      • OK: Maroon spot with purple border

      • Underoxidized: no purple border

    • In water:

      • OK: blue-black

      • Underoxidized: red-brown

The Nuclear Dyes

• Ehrlich hematoxylin

  • Hematoxylin

    • ___ oxidation

  • ___

  • ___

  • ___

  • ___

    • Aluminum is ___

  • ___

  • Used ___, rarely ___

The Nuclear Dyes

• Ehrlich hematoxylin

  • Hematoxylin

    • Physical or chemical oxidation

  • 95% alcohol

  • DI water

  • Glycerol

  • Ammonium/potassium aluminum sulfate

    • Aluminum is mordant

  • Glacial acetic acid

  • Used regressively, rarely progressively

The Nuclear Dyes

• Gill hematoxylin 1

  • ___

  • ___

    • ___ for hematoxylin

    • Prevents ___

  • ___

  • ___

    • oxidizer

  • ___

  • ___

  • ___ is stained blue

  • Hematoxylin only toxic if___ is used

  • Hematoxylin must be ___ before use or there will be ___in the section

    • ___ and ___ usually don't

    • ___ used to stop it

  • ___ solutions are fresher

    • ___ as it ages

    • Adding ___ turns it cherry red

    • ___ if overoxidized

  • ___ is more common than ___

The Nuclear Dyes

• Gill hematoxylin 1

  • DI water

  • Ethylene glycol

    • Solvent for hematoxylin

    • Prevents precipitate formation

  • Anhydrous hematoxylin

  • Sodium iodate

    • oxidizer

  • Aluminum sulfate

  • Glacial acetic acid

  • Mucin is stained blue

  • Hematoxylin only toxic if mercuric chloride is used

  • Hematoxylin must be filtered before use or there will be precipitate in the section

    • Mayer and Gill usually don't

    • Acetic acid used to stop it

  • Blue solutions are fresher

    • Redder as it ages

    • Adding glacial acetic acid turns it cherry red

    • Brown if overoxidized

  • Regressive is more common than progressive

The Nuclear Dyes

• Bluing:

  • Colors from ___ to ___

  • Stain insoluble in ___ and ___

  • Use ___ solutions to blue

    • ___

    • ___

    • ___

The Nuclear Dyes

• Bluing:

  • Colors from red to blue

  • Stain insoluble in water and alcohol

  • Use weak alkaline solutions to blue

    • Tap water

    • Lithium carbonate

    • Ammonium hydroxide

The Nuclear Dyes

• Bluing:

• Lithium carbonate solution (saturated)

  • ___

  • ___

  • it is ___

The Nuclear Dyes

• Bluing:

• Lithium carbonate solution (saturated)

  • Lithium carbonate

  • DI water

  • gentle

The Nuclear Dyes

• Bluing:

• 0.2% ammonia water solution

  • ___

  • ___

  • ___

The Nuclear Dyes

• Bluing:

• 0.2% ammonia water solution

  • Ammonium hydroxide

  • DI water

  • Fast but harsh

The Nuclear Dyes

• Bluing:

• Scott tap water substitute

  • ___

  • ___

  • ___

  • ___

The Nuclear Dyes

• Bluing:

• Scott tap water substitute

  • Magnesium sulfate

  • Sodium bicarbonate

  • Tap water

  • gentle

The Nuclear Dyes

• Weigert hematoxylin

  • ___

    • Mordant and oxidizer

  • ___

  • ___

  • ___

  • ___

  • Unstable

  • ___ in color

  • Used in ___ staining

The Nuclear Dyes

• Weigert hematoxylin

  • 29% ferric chloride

    • Mordant and oxidizer

  • DI water

  • Concentrated HCl

  • Hematoxylin

  • 95% alcohol

  • Unstable

  • Black in color

  • Used in trichrome staining

The Nuclear Dyes

Nuclear Dyes used as counterstains

• The point of counterstains is to ___

• ___ used for histochemistry

  • EX: ___ stain with___counterstain

• ___, ___, and ___ used as mordants

• ___ used for immunohistochemistry

  • EX: contrast with brown from___

  • EX: contrast with red from___

The Nuclear Dyes

Nuclear Dyes used as counterstains

• The point of counterstains is to produce contrast

• Red nuclear stains used for histochemistry

  • EX: blue perl iron stain with red nuclear counterstain

• Carmalum, brazalum, and aluminum salt used as mordants

• Blue nuclear stains used for immunohistochemistry

  • EX: contrast with brown from DAB IHC

  • EX: contrast with red from alkaline IHC

The Nuclear Dyes

Hematoxylin Substitutes

• ___, ___, ___, ___, ___

  • ___ is (+)

  • ___ is (-)

The Nuclear Dyes

Hematoxylin Substitutes

• Iron celestine blue, mordant blue 14, eriochrome cyanine R, mordant blue 3, solochrome cyanine R

  • Celestine blue is (+)

  • Eriochrome is (-)

The Nuclear Dyes

Hematoxylin Substitutes

• Celestine Blue

  • ___

  • ___

  • ___

  • ___

The Nuclear Dyes

Hematoxylin Substitutes

• Celestine Blue

  • Celestine Blue

  • Ferric ammonium sulfate

  • Glycerol

  • DI water

The Nuclear Dyes

Hematoxylin Substitutes

• Gallein Iron Hematoxylin

  • ___

  • ___

  • ___

  • ___

  • ___

  • ___

The Nuclear Dyes

Hematoxylin Substitutes

• Gallein Iron Hematoxylin

  • Gallein

  • Ethylene glycol

  • Absolute alcohol

  • DI Water

  • HCl

  • Ferric ammonium sulfate

The Nuclear Dyes

Plasma Stains

• Commonly ___ charge

• Bind to ___, ___,___

• ___ is most commonly used counterstain in routine staining

• Eosin is charged at pH___ but only stains below pH ___ due to ___

  • Not below pH ___, creates nonspecific binding and ___ sections

• 3 shades of pink

  • ___: deepest shade

    • ___ and ___ cells have a redder pink

  • ___: lightest OR intermediate shade

    • Lightest maybe due to F, P, or S

  • ___: lightest OR intermediate shade

    • Lightest maybe due to F, P, or S

  • Due to ___ differentiation

The Nuclear Dyes

Plasma Stains

• Commonly anionic (-) charge

• Bind to arginine, histidine, lysine

• Eosin is most commonly used counterstain in routine staining

• Eosin is charged at pH 7 but only stains below pH 6 due to protein IEP (+) charge

  • Not below pH 4, creates nonspecific binding and "muddy" sections

• 3 shades of pink

  • Red blood cells: deepest shade

    • Eosinophils and paneth cells have a redder pink

  • Collagen: lightest OR intermediate shade

    • Lightest maybe due to F, P, or S

  • Cytoplasm of muscle/epithelial cells: lightest OR intermediate shade

    • Lightest maybe due to F, P, or S

  • Due to graded alcohol differentiation

The Nuclear Dyes

Plasma Stains

• Eosin Solution

  • ___

  • ___

  • ___

  • pH should be ___-___

  • Very easy to ___

The Nuclear Dyes

Plasma Stains

• Eosin Solution

  • Eosin Y

  • 95% Ethanol

  • Glacial acetic acid

  • pH should be 4.6-5.0

  • Very easy to overstain

The Nuclear Dyes

Plasma Stains

• Eosin-phloxine B solution

  • ___

  • ___

  • ___

  • ___

  • Counterstaining takes___ and is ___

  • Collagen is colored ___

The Nuclear Dyes

Plasma Stains

• Eosin-phloxine B solution

  • Eosin Y

  • 95% Alcohol

  • Phloxine B

  • Glacial acetic acid

  • Counterstaining takes longer and is more expensive

  • Collagen is colored yellow

The Nuclear Dyes

H&E Staining

• Manual Progressive Staining Method

  • Used in ___ pathology

  • Mayer hematoxylin takes ___

  • 96% Harris hematoxylin/___ stains fast

  1. ___ x___ ___ min

  2. ___

  3. ___ x___

  4. ___ until ___

  5. ___ Hematoxylin ___ min OR ___ hematoxylin w ___ ___ min

  6. ___ x___

  7. ___ until ___

  8. ___ x___

  9. ___ OR ___ ___ min

  10. ___

  11. ___

  12. ___ x___

  13. ___ x___

The Nuclear Dyes

H&E Staining

• Manual Progressive Staining Method

  • Used in surgical pathology

  • Mayer hematoxylin takes too long

  • 96% Harris hematoxylin/glacial acetic acid stains fast

  1. Xylene x3 2 min

  2. Absolute alcohol

  3. 95% alcohol x2

  4. Tap water until clear

  5. Mayer Hematoxylin 15 min OR Harris hematoxylin w GAA 1-3 min

  6. Tap water x2

  7. Bluing until blue

  8. Tap water x2

  9. Eosin OR Eosin-phloxine 1-3 min

  10. 70% alcohol

  11. 95% alcohol

  12. Absolute alcohol x3

  13. Xylene x3

The Nuclear Dyes

H&E Staining

• Manual Regressive Staining Method

  1. ___ x___ ___ min

  2. ___

  3. ___ x___

  4. ___ until ___

  5. ___hematoxylin OR ___ hematoxylin with ___ ___min

  6. ___ x___

  7. ___ in ___

  8. ___until ___

  9. ___ until ___

  10. ___ OR ___ ___min

  11. ___

  12. ___

  13. ___ x___

  14. ___ x___

  • Be sure to not over- or under-___

The Nuclear Dyes

H&E Staining

• Manual Regressive Staining Method

  1. Xylene x3 2 min

  2. Absolute alcohol

  3. 95% alcohol x2

  4. Tap water until clear

  5. Delafield/Ehrlich hematoxylin OR Harris hematoxylin with no acid 10-15 min

  6. Tap water x2

  7. 1% HCl in 70% alcohol

  8. Running water until clear

  9. Bluing until blue

  10. Eosin OR Eosin-phloxine 1-3 min

  11. 70% alcohol

  12. 95% alcohol

  13. Absolute alcohol x3

  14. Xylene x3

  • Be sure to not over- or under- blue (differentiate)

The Nuclear Dyes

Automated H&E Staining

• Linear stainers

  • ___ or ___ of slides

  • Can only change solution time by ___

  • ___ for small, ___ for big

  • ___ staining used

• Robotic/Batch stainers

  • ___ slides

  • Completely computerized

  • ___ staining used

• Secret third type

  • ___ or ___ of slides

  • Use ___ for every step, prevent ___

• Upkeep

  • Keep ___

  • Filter and rotate hematoxylin ___

  • Change all solutions except ___ daily

  • Change ___weekly

The Nuclear Dyes

Automated H&E Staining

• Linear stainers

  • Individual or small groups of slides

  • Can only change solution time by adding more containers

  • 30 sec for small, 1 min for big

  • Progressive staining used

• Robotic/Batch stainers

  • Baskets of 20 slides

  • Completely computerized

  • Progressive/regressive staining used

• Secret third type

  • Individual or baskets

  • Use fresh reagents for every step, prevent cross contamination

• Upkeep

  • Keep containers filled

  • Filter and rotate hematoxylin daily

  • Change all solutions except H&E daily

  • Change E weekly

The Nuclear Dyes

Notes on the Results of H&E Staining

• ___ appear closed or ___

• ___ appear open or ___

• Good H&E stains are described as ___

• Balanced slide is referred to as:

  • ___ intensity nuclear stain, showing that ___ was done correctly

  • ___ intensity counterstain, showing that ___ was done correctly

  • No ___

  • ___ staining

  • Good contrast between ___ and ___

The Nuclear Dyes

Notes on the Results of H&E Staining

• Lymphocytes appear closed or hyperchromatic

• Epithelial cells appear open or vesicular

• Good H&E stains are described as crisp

• Balanced slide is referred to as:

  • Medium intensity nuclear stain, showing that bluing was done correctly

  • Medium intensity counterstain, showing that bluing was done correctly

  • No stain precipitate

  • Even staining

  • Good contrast between nuclear and counterstain

The Nuclear Dyes

The H&E Control

• Common tissues used for QC are ___, ___, ___, ___

• One block with ___ is used as well

• ___ slides can also be used for QC

The Nuclear Dyes

The H&E Control

• Common tissues used for QC are appendix, prostate, uterus, placenta

• One block with multiple types of tissue on it is used as well

• Previous patient slides can also be used for QC

The Nuclear Dyes

Hints for achieving excellent H&E staining

• ___ using microscope

  • ___shades of eosin

  • ___borders

  • Correct ___ and ___

• ___ BEFORE running the patient slides

• Keep sections ___during staining

• Keep solutions covered to ___

  • ___ if precipitate forms

• Make a ___

• Wash with tap water if using ___as a ___agent

• Don’t pass thru the differentiating solutions ___

• Adjust staining times for different ___

• Check solution compatibility if using ___

• Use ___ before and after using  ___

  • ___ interfere with staining

• Rinse ___containers before use

  • ___ from soap can bind to hematein

• Standard section ___ guidelines

• Pathologists also have ___

The Nuclear Dyes

Hints for achieving excellent H&E staining

• Slide QC using microscope

  • 3 shades of eosin

  • Crisp borders

  • Correct intensity and contrast

• Run a QC slide BEFORE running the patient slides

• Keep sections moist during staining

• Keep solutions covered to prevent evaporation

  • Filter solution if precipitate forms

• Make a routine schedule

• Wash with tap water if using ammonia as a bluing agent

• Don’t pass thru the differentiating solutions quickly

• Adjust staining times for different fixatives

• Check solution compatibility if using a xylene substitute

• Use DI water before and after using hematoxylin

  • Ions interfere with staining

• Rinse hematoxylin containers before use

  • Phosphates from soap can bind to hematein

• Standard section thickness guidelines

• Pathologists also have personal preferences

The Nuclear Dyes

Restoring Tissue Basophilia

• Loss of tissue basophilia: ___

• Caused by ___, ___, ___

  • Loss of staining via ___ cannot be rectified

  • ___hematoxylin is best for rectifying overfixed tissue stains

• Method 1

  • Strictly for overexposure to ___

  1. ___ for ___

  2. ___ for ___

  3. Stain

• Method 2

  1. ___ for ___

  2. ___ for___

  3. Stain

• Method 3

  1. ___ for ___

  2. ___ x___

  3. Stain

The Nuclear Dyes

Restoring Tissue Basophilia

• Loss of tissue basophilia: loss of hematoxylin stain

• Caused by overfixation, inadequate dehydration, overdecalcification

  • Loss of staining via overdecalcification cannot be rectified

  • Weigert hematoxylin is best for rectifying overfixed tissue stains

• Method 1

  • Strictly for overexposure to Bouins

  1. 5% lithium carbonate for 1 hour

  2. Running tap water for 10 min

  3. Stain

• Method 2

  1. 5% sodium bicarbonate for 3 hours

  2. Tap water for 5 min

  3. Stain

• Method 3

  1. 5% periodic acid for 30 min

  2. DI water x3

  3. Stain

Frozen Section Staining

Fixative

• ___

• ___

Frozen Section Staining

Fixative

• 95% alcohol

• Concentrated glacial acetic acid

Frozen Section Staining

Procedure

1. Cut section, place on slide, and ___ for ___

2. ___

3. ---

   for ___

4. ___

5. ___ until ___

6. ___

7. ___

8. ___

9. ___

10. ___ x___

11. Mount with ___

Frozen Section Staining

Procedure

1. Cut section, place on slide, and fix for 20 sec

2. Running tap water

3. Acidified harris hematoxylin 1 min

4. Running tap water

5. Bluing until blue

6. Running tap water

7. Eosin

8. 95% alcohol

9. Absolute alcohol

10. Xylene x3

11. Mount with resin

Frozen Section Staining

Notes on frozen section staining

• ___ used as a fixative as well

• Hematoxylin changed ___ or ___ minimum

  • ___ is reducing agent

• ___ and ___ solutions are used for ___ stains on ___sections

Frozen Section Staining

Notes on frozen section staining

• Alcoholic formalin used as a fixative as well

• Hematoxylin changed daily or biweekly minimum

  • Formaldehyde is reducing agent

• Toluidine blue O and polychrome solutions are used for semipermanent stains on unfixed sections

Troubleshooting the H&E Stain

Incomplete deparaffination

• Looks like ___ or ___

• Caused by

  • ___

  • ___ from processing

  • Not enough ___

  • Contaminated ___

• Rectified by

  • ___to remove ___

Troubleshooting the H&E Stain

Incomplete deparaffination

• Looks like white spots or spotty staining

• Caused by

  • Incomplete drying

  • Water in tissue from processing

  • Not enough clearing

  • Contaminated deparaffination solutions

• Rectified by

  • Alcohol to remove water

Troubleshooting the H&E Stain

Nuclear staining is not crisp

• Looks like nuclei are ___ or ___

• Caused by

  • ___

  • Too much ___ during P&E

• Rectified by

  • Don’t put tissues in ___ for too long

  • Slides ___ to dry as ___ and ___ as possible

Troubleshooting the H&E Stain

Nuclear staining is not crisp

• Looks like nuclei are muddy or smudgy

• Caused by

  • Poor fixation

  • Too much heat during P&E

• Rectified by

  • Don’t put tissues in paraffin for too long

  • Slides stood on end to dry as quickly and warmly as possible

Troubleshooting the H&E Stain

Nuclear Stain is too pale

• Caused by

  • Not enough time in ___

  • Using ___ hematoxylin

  • Over ___

  • Over ___

  • Inadequate ___

• Rectified by:

  • See "___"

Troubleshooting the H&E Stain

Nuclear Stain is too pale

• Caused by

  • Not enough time in hematoxylin

  • Using overoxidized hematoxylin

  • Over differentiating

  • Over decalcification

  • Inadequate dehydration

• Rectified by:

  • See "restoring tissue basophilia"

Troubleshooting the H&E Stain

Nuclear stain is too dark

• Caused by:

  • Too much time in ___

  • Under ___

  • Sections are ___

  • Incorrect ___ of hematoxylin

Troubleshooting the H&E Stain

Nuclear stain is too dark

• Caused by:

  • Too much time in hematoxylin

  • Under differentiating

  • Sections are too thick

  • Incorrect pH of hematoxylin

Troubleshooting the H&E Stain

Nuclear Bubbling

• Caused by:

  • Using ___ as a fixative

  • ___ fixation time

    •  ___ for biopsies

  • ___ while drying

  • Sections can't ___

Troubleshooting the H&E Stain

Nuclear Bubbling

• Caused by:

  • Using NBF as a fixative

  • Too little fixation time

    • Minimum of 6-8 hours for biopsies

  • Too much heat while drying

  • Sections can't drain all the water

Troubleshooting the H&E Stain

Red or red-brown nuclei

• Caused by:

  • ___ breaking down

  • Improper ___

Troubleshooting the H&E Stain

Red or red-brown nuclei

• Caused by:

  • Hematoxylin breaking down

  • Improper bluing

Troubleshooting the H&E Stain

Cytoplasmic Stain is too light

• Caused by:

  • pH is ___

    • Due to ___

  • Sections are ___

  • Over___

• Rectified by:

  • Check pH and Adjust with ___

    • ___-___ is ideal

Troubleshooting the H&E Stain

Cytoplasmic Stain is too light

• Caused by:

  • pH is above 5

    • Due to bluing carryover

  • Sections are too thin

  • Overdehydrated

• Rectified by:

  • Check pH and Adjust with acetic acid

    • 4.1-4.5 is ideal

Troubleshooting the H&E Stain

Cytoplasmic stain is too dark

• Nuclei look ___

• Rectified by:

  • Dilute ___ if necessary

  • Ensure slides arent left in ___ for too long

  • Make sure sections are ___

  • Enough ___ to allow for ___

Troubleshooting the H&E Stain

Cytoplasmic stain is too dark

• Nuclei look red-purple

• Rectified by:

  • Dilute eosin if necessary

  • Ensure slides arent left in eosin for too long

  • Make sure sections are thick/thin enough

  • Enough dehydration to allow for differentiation

Troubleshooting the H&E Stain

Eosin not properly differentiated

• Rectified by:

  • Complete ___

  • Enough ___ and ___ during P&E

  • Enough ___ to allow for ___

  • Correct___ of eosin

Troubleshooting the H&E Stain

Eosin not properly differentiated

• Rectified by:

  • Complete fixation

  • Enough dehydration and clearing during P&E

  • Enough dehydration to allow for differentiation

  • Correct pH of eosin

Troubleshooting the H&E Stain

Blue-black precipitate on top of sections

• Caused by:

  • ___ allowed to form over ___

• Rectified by:

  • ___ hematoxylin daily

Troubleshooting the H&E Stain

Blue-black precipitate on top of sections

• Caused by:

  • Metallic sheen allowed to form over hematoxylin

• Rectified by:

  • Filtering hematoxylin daily

Troubleshooting the H&E Stain

Water & Slides turn milky after being placed in water after deparaffination

• Caused by ___ on slide

• Rectified by changing out ___

Troubleshooting the H&E Stain

Water & Slides turn milky after being placed in water after deparaffination

• Caused by xylene on slide

• Rectified by changing out alcohols

Troubleshooting the H&E Stain

Slides are hazy or milky in xylene

• Caused by ___ on slide

• Rectified by changing out ___ and ___

Troubleshooting the H&E Stain

Slides are hazy or milky in xylene

• Caused by water on slide

• Rectified by changing out alcohols and xylenes

Troubleshooting the H&E Stain

Uneven H&E staining

• Caused by:

  • Not enough ___

  • Sections too ___

  • ___ in paraffin

  • ___ in alcohol

• Rectified by:

  • Change from ___ to ___

Troubleshooting the H&E Stain

Uneven H&E staining

• Caused by:

  • Not enough solution in container

  • Sections too thick/thin

  • Water/alcohol in paraffin

  • Water in alcohol

• Rectified by:

  • Change from xylene to toluene

Troubleshooting the H&E Stain

Dark basophilic staining of nuclei and cytoplasm

• Caused by ___ and ___

• Rectified by ___

Troubleshooting the H&E Stain

Dark basophilic staining of nuclei and cytoplasm

• Caused by laser and electrocautery

• Rectified by nothing

Troubleshooting the H&E Stain

Poor contrast between nucleus and cytoplasm

• Looks like:

  • Nuclear stain is ___ to contrast

  • Cytoplasm is ___ and masks ___

  • Nuclear stain is too ___ for cytoplasm stain

  • Cytoplasm stain is too ___ for nuclear stain

• Rectified by:

  • Find out whether problem is ___ or ___ stain

  • Check___ of stains

  • Check for ___

Troubleshooting the H&E Stain

Poor contrast between nucleus and cytoplasm

• Looks like:

  • Nuclear stain is too pale to contrast

  • Cytoplasm is overstained and masks nuclei

  • Nuclear stain is too dark for cytoplasm stain

  • Cytoplasm stain is too pale for nuclear stain

• Rectified by:

  • Find out whether problem is nuclear or cytoplasm stain

  • Check pH of stains

  • Check for contaminants