Nuclear and Cytoplasmic Staining CH 6 Flashcards
1
Q
Ultra structure of the cell
A
- Nucleus
- Cytoplasm
2
Q
Nucleus contains..
A
- Nuclear Membrane
- Nuclear Pores
- Nucleolus
- Chromatin
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.
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
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!
6
Q
Cytoplasm contents..
A
- Plasmalemma
- Mitochondria
- Ribosomes
- Endoplasmic Reticulum
- Golgi Apparatus
- Centriole
- Lysosome
7
Q
Plasmalemma
A
- Plasma membrane
- VERY important to antigen-antibody reactions
- Rarely seen on H&E stained tissue
8
Q
Ribosomes
A
- Site of protein synthesis
- Responsible for the bluish color in cytoplasm in come cells
8
Q
Mitochondria
A
- Produce energy for the cell, and is membrane bound
- Not seen on H&E stained sections
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
10
Q
Golgi Apparatus
A
- Packaging department of cell
- Not normally stained with H&E
11
Q
Centriole
A
- Used for spindle formation in cell division
- Can not be seen on H&E stained sections
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
13
Q
Staining Mechanisms
◦ Nuclear
◦ Cytoplasmic
A
◦ Nuclear
◦ Cytoplasmic
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
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
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
16
Q
Overview of cell charged structures
A
Cytoplasm: positive
Nucleus: negative
Rough ER: negative
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
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
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
19
Q
Staining Results:
A
cytoplasm: positive, pink
nucleus: negative, dark blue
rough ER: negative, dark blue
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)
21
Q
Factors Affecting Dyes
A
- pH
- Temp
- Concentration
- Salts/Ions
- Fixation
22
Dyes
- 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
Dyes
- 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
Differentiation
- Progressive and regressive staining procedures:
- 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
Differentiation
◦ Mordants
- 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
Differentiation
- Basic/cationic dyes are differentiated by weak acid solutions (aluminum hematoxylins with dilute HCL)
- 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
Nuclear Dyes
◦ Hematoxylin!
- 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
Nuclear Dyes
◦ Iron Hematoxylins
- 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
Nuclear Dyes
- 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
Cytoplasmic Dyes
- Eosin is most common and widely used
- 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
Hematoxylins
- Harris Hematoxylin
- Oxidizer: Mercuric oxide
- Mordant: Ammonium aluminum sulfate
- Used: Regressive
30
Harris Hematoxylin
- Traditionally used Mercuric oxide, today uses
Sodium Iodate 2.5g
- Traditionally used Mercuric oxide, today uses Sodium Iodate 2.5g
- Ammonium aluminum sulfate 100g
- Absolute ethyl alcohol 50mL
- Distilled water 1L
- Hematoxylin 5g
31
Delafield Hematoxylin
- 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
Hematoxylins
- Delafield Hematoxylin
- 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
Mayer Hematoxylin contents..
- Hematoxylin 1g
- Distilled water 1L
- Sodium Iodate 0.2g
- Ammonium or potassium aluminum sulfate 50g
- Citric acid 1g
- Chloral hydrate 50g
34
Hematoxylins
- Mayer Hematoxylin
- 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
Ehrlich Hematoxylin contents..
- Hematoxylin 2g
- 95% Alcohol 100mL
- Distilled Water 100mL
- Ammonium or potassium aluminum sulfate 3g
- Glacial acetic acid 10mL
36
Gill Hematoxylin contents...
- Distilled water 730mL
- Ethylene glycol 250mL
- Hematoxylin, anhydrous 2g
- Sodium iodate 17.6g
- Glacial acetic acid 20mL
36
Hematoxylins
- Ehrlich Hematoxylin
- Oxidizer: sunlight and air
- Mordant: Ammonium or Potassium aluminum sulfate
- Used: Regressive / Progressive
- Sodium iodate can be added for immediate ripening
37
Hematoxylins
- Gill Hematoxylin
- Oxidizer: Sodium iodate
- Mordant: Aluminum sulfate
- Used: Regressive
- Goblet cells will be stained
38
Weigert Hematoxylin contents..
- 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
Hematoxylins
- Weigert Hematoxylin
- Oxidizer & Mordant: Ferric chloride
- Used: Regressive
- Resists decolorization from subsequent acidic staining solutions.
40
Nuclei Staining Alternatives
- Celestine Blue
◦ Also:
- Gallein
- Safranin,
- Nuclear-fast Red
- Methylene blue
- Thionin
- Toluidine blue O
41
Plasma Stains
- 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
Eosin
- Will produce three (3) shades of pink
◦ 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
Routine Staining
- Troubleshooting
- Problem: White spots are seen in the section after the deparaffinization step.
- 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
Routine Staining
- Troubleshooting
◦ Problem: Nuclei are too pale.
- Problem: Nuclei are too pale.
◦ Cause:
- Section was not stained long enough in hematoxylin OR,
- Hematoxylin is overoxidized OR,
- Differentiation was too long
45
Routine Staining
- Troubleshooting
◦ Problem: Nuclei are overstained.
◦ Problem: Nuclei are overstained.
◦ Cause:
- Section was stained long enough in hematoxylin OR,
- Sections are too thick OR,
- Differentiation was too short
46
Routine Staining
- Troubleshooting
◦ Problem: Red or Brown nuclei.
◦ Problem: Red or Brown nuclei.
◦ Cause:
- Hematoxylin is breaking down OR,
- Sections were not blued sufficiently
47
Routine Staining
- Troubleshooting
◦ Problem: Pale staining with eosin.
◦ 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
Routine Staining
- Troubleshooting
◦ Problem: Cytoplasm is overstained and differentiation is poor.
◦ 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
Routine Staining
- Troubleshooting
◦ Problem: Blue-black precipitate on top of the section.
◦ 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
Routine Staining
- Troubleshooting
◦ Problem: Water bubble are seen microscopically in the stained sections.
◦ Problem: Water bubble are seen microscopically in the stained sections.
◦ Cause:
- Sections were not completely dehydrated and water is present in mounting medium.
51
Routine Staining
- Troubleshooting
◦ Problem: Water and slides turn milky when slides are placed in water following the rehydrating alcohols.
◦ 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
Troubleshooting
◦ Problem: Hazy blue nuclei.
◦ 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
Routine Staining
- Troubleshooting
◦ Problem: Brown stippling resembling pigment and glossy black nuclei are in section.
◦ Problem: Brown stippling resembling pigment and glossy black nuclei are in section.
◦ Cause:
- Section was allowed to air dry before coverslipping.
