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Microtechniques > Nuclear and Cytoplasmic Staining CH 6 > Flashcards

Nuclear and Cytoplasmic Staining CH 6 Flashcards

1
Q

Ultra structure of the cell

A
  • Nucleus
  • Cytoplasm
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2
Q

Nucleus contains..

A
  • Nuclear Membrane
  • Nuclear Pores
  • Nucleolus
  • Chromatin
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3
Q

Nucleus

A
  • Appearance can differ from whether the cell is resting or dividing (mitosis).
  • Resting nucleus is sometimes called an interphase nucleus because it is between
    two stages of cell division.
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4
Q

◦ Nuclear Membrane
◦ Nuclear Pores
◦ Nucleolus

A

◦ Nuclear Membrane
- Usually stained crisp dark blue with aluminum hematoxylins

◦ Nuclear Pores
- Can only be seen with electron microscope

◦ Nucleolus
- Dark round mass made of 80 90% protein.
- Contains most of the ribosomal RNA

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5
Q

Chromatin
◦ Two distinct types of chromatin:
1.) Heterochromatin
2.) Euchromatin

A
  • Heterochromatin refers to the stainable type (basophilic) that contains dense regions of
    chromosomes.
  • Euchromatin does not stain well because the DNA
    is still genetically active.
  • Histones are also basic proteins that may affect
    nuclear staining.
  • Chromatin patterns can be used to determine is a
    tissue section has been overstained!
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6
Q

Cytoplasm contents..

A
  • Plasmalemma
  • Mitochondria
  • Ribosomes
  • Endoplasmic Reticulum
  • Golgi Apparatus
  • Centriole
  • Lysosome
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7
Q

Plasmalemma

A
  • Plasma membrane
  • VERY important to antigen-antibody reactions
  • Rarely seen on H&E stained tissue
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8
Q

Ribosomes

A
  • Site of protein synthesis
  • Responsible for the bluish color in cytoplasm in come cells
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8
Q

Mitochondria

A
  • Produce energy for the cell, and is membrane bound
  • Not seen on H&E stained sections
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9
Q

Rough Endoplasmic Reticulum

A
  • These organelles have many ribosomes, and are responsible for the basophilic nature of some cells
  • Plasma cells and pancreatic acinar cells are examples of cells with high amounts of rough E.R. and are basophilic
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10
Q

Golgi Apparatus

A
  • Packaging department of cell
  • Not normally stained with H&E
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11
Q

Centriole

A
  • Used for spindle formation in cell division
  • Can not be seen on H&E stained sections
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12
Q

Lysosomes

A
  • Used to “digest” or destroy compounds
  • Can also be used to destroy bad or unhealthy cells, which creates indigestible compounds which can build up
  • These compounds are called residual bodies, and can build up in tissues which is known as lipofuscin or “wear-and-tear”
    pigment
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13
Q

Staining Mechanisms
◦ Nuclear
◦ Cytoplasmic

A

◦ Nuclear
◦ Cytoplasmic

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14
Q

Staining Mechanisms
◦ Most stains involve both physical & chemical reactions

A
  • Dye is bound to tissue primarily by either:
  • Ionic bonding
  • Hydrogen bonding
  • Covalent bonding
  • Van der Waals forces
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14
Q

◦ Basic:
◦ Acidic:
◦ Cationic:
◦ Anionic:
◦ Basophilic:
◦ Acidophilic:

A
  • Basic: substance with net positive charge
  • Acidic: substance with net negative charges
  • Cationic: substance with net positive charges
  • Anionic: substance with net negative charges
  • Basophilic: substances that attracts basic/cationic dyes
  • Acidophilic: substance that attracts acidic/anionic dyes
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15
Q

Nuclear Staining Mechanisms
◦ Not fully understood
◦ Two (2) possible ways:

A

1.) Staining is done with a basic (cationic or + charged) dye

2.) Staining is done with dyes combined with or
followed by metal mordants

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16
Q

Overview of cell charged structures

A

Cytoplasm: positive
Nucleus: negative
Rough ER: negative

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17
Q

Cytoplasmic Staining Mechanisms

A
  • The terminal ends of proteins have an amino (NH2) or a carboxyl (COOH) group
  • These two groups can make a protein positively or negatively charged.
  • The charge is pH dependent, and because proteins can carry either a + or – charge, they are said to be amphoteric
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17
Q

Cytoplasmic Staining Mechanisms

A
  • IEP: for proteins, is around pH 6.
  • Below IEP (pH 6) net charge on
    cytoplasmic proteins is positive, and attracts anionic dyes.
  • Above IEP (pH 6) net charge on
    cytoplasmic proteins is negative and attracts cationic dyes
  • This means, if sections are placed in a substance that’s pH is below 6, it will develop a positive charge, and react with a anionic dye such as eosin
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18
Q

Cytoplasmic Staining Mechanisms

A
  • Net charge of a substance determines where is will migrate on an electrical field
  • Net positive charges migrate towards cathodes (-)
  • Net negative charges migrate towards anodes (+)
  • The ISOELECTRIC POINT is where the positive and negative charges are equal, and
    there is no migration. Basically, the IEP is the pH where a protein carries no electrical charge
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19
Q

Staining Results:

A

cytoplasm: positive, pink
nucleus: negative, dark blue
rough ER: negative, dark blue

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20
Q

Terms of Dyes

A
  • Chromophores are responsible for giving dyes their color by absorbing certain wavelengths of visible light and transmitting
    or reflecting others.
  • Chromogen: substance that can be converted into a color pigment or dye
  • Auxochromes are used to modify the ability of a chromophore to absorb light. Used to intensify the color
  • Absorption: physically staining where dye is dissolved into stained element (lipids)
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21
Q

Factors Affecting Dyes

A
  • pH
  • Temp
  • Concentration
  • Salts/Ions
  • Fixation
22
Q

Dyes

A
  • pH: pH of solution can determine what tissue components stain with a specific dye
  • Temp: In general, increased in temperature will increase rate at which dye is diffused into
    tissue
  • Concentration: dyes usually act faster with higher concentrations
  • Salts/Ions: can increase or decrease rate of stain. Is believed salts compete for binding sites
23
Q

Dyes

A
  • Fixation: can affect rate of stain in many ways
  • Fixative can react with chemical groups making them unavailable for subsequent staining
  • Formalin reacts with amino group (NH2), as does eosin. So formalin fixed tissue will bind
    less eosin
  • Potassium Dichromate reacts with carboxyl groups (COOH) and will take up more eosin
    and less hematoxylin
  • Zenkers and Bouin are acidic and will mask nuclear stains if overexposed
24
Q

Differentiation
- Progressive and regressive staining procedures:

A
  • Progressive and regressive staining procedures:
    1.) Progressive: once desired color intensity of stain is reached, reaction is stopped

2.) Regressive: sections are overstained, and then
differentiated or decolorized until staining is completed

25
Q

 Differentiation
◦ Mordants

A
  • Mordants: substances or metals that act as a link between dye and tissue
  • Mordants bind with the dye to make a “dye lake” where the tissue sections can be readily
    stained, usually are basic in action (hematoxylins)
26
Q

Differentiation
- Basic/cationic dyes are differentiated by weak acid solutions (aluminum hematoxylins with dilute HCL)

A
  • Basic/cationic dyes are differentiated by weak acid solutions (aluminum hematoxylins with dilute HCL)
  • Acidic/anionic dyes are differentiated by weak alkali or basic solutions (eosins with
    dilute ammonium hydroxide)
  • Excess mordant will bind the dye and remove it from tissue (iron hematoxylins with excess
    iron)
  • Oxidizing solutions can also take dye out of the tissue to help differentiate (potassium
    ferricyanide in Weil stain)
27
Q

Nuclear Dyes
◦ Hematoxylin!

A
  • Most widely used, comes in many different forms
  • Extracted from the logwood tree
  • Hematoxylin itself is NOT a dye, the oxidized form hematein is
  • MUST be oxidized to be used, oxidizing agents commonly used are: sodium iodate, mercuric
    chloride, and potassium permanganate.
  • Atmospheric oxygen also slowly oxidizes hemtoxylin!
28
Q

Nuclear Dyes
◦ Iron Hematoxylins

A
  • Have the ability to use the mordant as an oxidizer
  • Most commonly used is Weigerts Hematoxylin
  • Has ability to resist decolorization with acids
  • Ferric Chloride is normally used as mordant AND oxidizer
  • Celestine Blue is used progressively and uses Iron as the mordant
    *Is a substitue for hematoxylin and gives identical results
28
Q

Nuclear Dyes

A
  • Hematein has little affinity for tissue
  • Must be paired with the appropriate mordants or metal
  • Alum hematoxylins have potassium aluminum sulfate, ammonium aluminum
    sulfate, ferric ammonium sulfate, or chromium potassium sulfate
  • Gills hematoxylin uses a single sulfate mordant; aluminum sulfate
29
Q

Cytoplasmic Dyes
- Eosin is most common and widely used

A
  • Eosin is most common and widely used
  • IEP of proteins is 6, so must be kept at a pH below 6
  • If pH drops below 4 however, the amount of charged dye will drop and not stain
  • Eosin is best kept at pH range 4.6-5
  • 3 shades of pink: RBC’s, collagen, and cytoplasm
  • Eosin phloxine B is another type commonly used that
    the pink shades are more vivid, however it is easy to overstain
30
Q

Hematoxylins
- Harris Hematoxylin

A
  • Oxidizer: Mercuric oxide
  • Mordant: Ammonium aluminum sulfate
  • Used: Regressive
30
Q

Harris Hematoxylin
- Traditionally used Mercuric oxide, today uses
Sodium Iodate 2.5g

A
  • Traditionally used Mercuric oxide, today uses Sodium Iodate 2.5g
  • Ammonium aluminum sulfate 100g
  • Absolute ethyl alcohol 50mL
  • Distilled water 1L
  • Hematoxylin 5g
31
Q

Delafield Hematoxylin

A
  • Solution A: ammonium aluminum sulfate 180g, distilled water 1L (saturated ammonium sulfate)
  • Solution B: hematoxylin 4g, 95% alcohol 25mL,
  • Combine 400mL Solution A and 25mL solution B
  • Then add 100mL glycerol
  • Ethyl Alcohol 95% 100mL
32
Q

Hematoxylins
- Delafield Hematoxylin

A
  • Oxidizer: sunlight and air
  • Mordant: Ammonium aluminum sulfate
  • Used: Regressive
  • Glycerol aids in overoxidation
  • Prepared by combining two solutions
    1.) Solution A: Oxidizer, et al
    2.) Solution B: Hematoxylin, et al
33
Q

Mayer Hematoxylin contents..

A
  • Hematoxylin 1g
  • Distilled water 1L
  • Sodium Iodate 0.2g
  • Ammonium or potassium aluminum sulfate 50g
  • Citric acid 1g
  • Chloral hydrate 50g
34
Q

Hematoxylins
- Mayer Hematoxylin

A
  • Oxidizer: Sodium iodate
  • Mordant: Ammonium or Potassium aluminum sulfate
  • Used: Progressive
  • Includes citric acid and choral hydrate to adjust pH and prevent “surface scum” precipitate.
  • Used progressively, difficult to overstain with
  • Used for IHC stains involving AEC as chromogen because it does not contain alcohol
35
Q

Ehrlich Hematoxylin contents..

A
  • Hematoxylin 2g
  • 95% Alcohol 100mL
  • Distilled Water 100mL
  • Ammonium or potassium aluminum sulfate 3g
  • Glacial acetic acid 10mL
36
Q

Gill Hematoxylin contents…

A
  • Distilled water 730mL
  • Ethylene glycol 250mL
  • Hematoxylin, anhydrous 2g
  • Sodium iodate 17.6g
  • Glacial acetic acid 20mL
36
Q

Hematoxylins
- Ehrlich Hematoxylin

A
  • Oxidizer: sunlight and air
  • Mordant: Ammonium or Potassium aluminum sulfate
  • Used: Regressive / Progressive
  • Sodium iodate can be added for immediate ripening
37
Q

Hematoxylins
- Gill Hematoxylin

A
  • Oxidizer: Sodium iodate
  • Mordant: Aluminum sulfate
  • Used: Regressive
  • Goblet cells will be stained
38
Q

Weigert Hematoxylin contents..

A
  • Solutions A: Ferric chloride 4mL, distilled water 95mL, hydrochloric acid 1mL.
  • Solution B: hematoxylin 1g, 95% alcohol 100mL
  • Working solution: mix equal parts of A and B. Can be used for no more than 3 days. Prepare fresh
39
Q

Hematoxylins
- Weigert Hematoxylin

A
  • Oxidizer & Mordant: Ferric chloride
  • Used: Regressive
  • Resists decolorization from subsequent acidic staining solutions.
40
Q

Nuclei Staining Alternatives

A
  • Celestine Blue

◦ Also:
- Gallein
- Safranin,
- Nuclear-fast Red
- Methylene blue
- Thionin
- Toluidine blue O

41
Q

Plasma Stains

A
  • Eosin is the most widely used counterstain in routing staining.
  • Remember! To stain for cytoplasm, pH must be below 6 to develop net positive charge on proteins.
  • Best staining with eosin will occur at a pH of approximately 4.6 to 5.
42
Q

Eosin
- Will produce three (3) shades of pink

A

◦ Will produce three (3) shades of pink:

1.) Erythrocytes: bright pink/ orange pink

2.) Collagen: light pink

3.) Cytoplasm of muscle or epithelial cells: dark pink

◦ When using alcohol based eosin, go directly into alcohols when running down to xylene for coverslipping.

43
Q

Routine Staining
- Troubleshooting
- Problem: White spots are seen in the section after the deparaffinization step.

A
  • Problem: White spots are seen in the section after the deparaffinization step.

◦ Cause:
- Section was not dried property prior to deparaffinization OR
*slide did not remain in xylene long enough to remove paraffin.

44
Q

Routine Staining
- Troubleshooting
◦ Problem: Nuclei are too pale.

A
  • Problem: Nuclei are too pale.

◦ Cause:
- Section was not stained long enough in hematoxylin OR,

  • Hematoxylin is overoxidized OR,
  • Differentiation was too long
45
Q

Routine Staining
- Troubleshooting
◦ Problem: Nuclei are overstained.

A

◦ Problem: Nuclei are overstained.

◦ Cause:
- Section was stained long enough in hematoxylin OR,

  • Sections are too thick OR,
  • Differentiation was too short
46
Q

Routine Staining
- Troubleshooting
◦ Problem: Red or Brown nuclei.

A

◦ Problem: Red or Brown nuclei.

◦ Cause:

  • Hematoxylin is breaking down OR,
  • Sections were not blued sufficiently
47
Q

Routine Staining
- Troubleshooting
◦ Problem: Pale staining with eosin.

A

◦ Problem: Pale staining with eosin.

◦ Cause:
- pH is above 5, OR,

  • Possible carryover from bluing reagent, OR
  • Sections are too thin, OR
  • Pale staining with eosin
48
Q

Routine Staining
- Troubleshooting
◦ Problem: Cytoplasm is overstained and differentiation is poor.

A

◦ Problem: Cytoplasm is overstained and differentiation is poor.

◦ Cause:
- Eosin is too concentrate, OR

  • Sections were stained for too long, OR
  • Sections were passed through alcohols too quickly for good differentiation.
49
Q

Routine Staining
- Troubleshooting
◦ Problem: Blue-black precipitate on top of the section.

A

◦ Problem: Blue-black precipitate on top of the section.

◦ Cause:
- Metallic sheen that develops on surface of hematoxylin has been “picked up” on slide

50
Q

Routine Staining
- Troubleshooting
◦ Problem: Water bubble are seen microscopically in the stained sections.

A

◦ Problem: Water bubble are seen microscopically in the stained sections.

◦ Cause:
- Sections were not completely dehydrated and water is present in mounting medium.

51
Q

Routine Staining
- Troubleshooting
◦ Problem: Water and slides turn milky when slides are placed in water following the rehydrating alcohols.

A

◦ Problem: Water and slides turn milky when slides are placed in water following the rehydrating alcohols.

◦ Cause:
- Xylene was not completely removed by alcohols.

52
Q

Troubleshooting
◦ Problem: Hazy blue nuclei.

A

◦ Problem: Hazy blue nuclei.

◦ Cause:
- Use of too much heat on tissue processor, OR

  • Holding tissue in hot paraffin for too long, OR
  • Too short of fixation followed by dehydration directly into alcohols.
53
Q

Routine Staining
- Troubleshooting
◦ Problem: Brown stippling resembling pigment and glossy black nuclei are in section.

A

◦ Problem: Brown stippling resembling pigment and glossy black nuclei are in section.

◦ Cause:
- Section was allowed to air dry before coverslipping.

Microtechniques (10 decks)

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