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Histo 1 > Histology: Stains and Cell Ultrastructure > Flashcards

Histology: Stains and Cell Ultrastructure Flashcards

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1
Q

Define: Histology

A

The study of functional tissue

aka Microanatomy

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2
Q

Nomarski (DIC)

A

pseudo 3D organelles

not used much with tissues

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3
Q

Giemsa Staining

A

common in research labs

uses eosin and methylene blue to stain cells

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4
Q

What is the most common stain?

A

Hematoxylin (blue-purple) and Eosin (orange-pink)

(H&E)

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5
Q

Basophilic structures

A
  • Stain with hematoxylin
  • ribosomes, nucleic acids, nucleus, rough er
  • Basophilic = Blue
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6
Q

Eosinophilic Structures

A

Acidophilic

stain with Eosin (orange/pink)

mitochondria, lysosomes, cytoplasm

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7
Q

What is normally lost during fixation?

A

Lipids/Carbohydrates

Ground substance (water, salt)

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8
Q

What are tissues normally processed in?

A

formaldehyde → it crosslinks proteins

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9
Q

How do you visualize cells in an H&E Stain?

A

Cytoplasm → pink

nucleus → purple

empty space → where glycogen and other carbohydrates were lost

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10
Q

Cresyl Violet (RNA)

A

Stains neurons, rough ER, and polysomes really well

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11
Q

Periodic Acid Schiff (PAS)

A

Stains carbohydrates (basement membrane, mucus) (magenta)

Schiffs reagent stains nuclei (DNA)

counterstain with Fast Green FCF (cytoplasm, collagen)

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12
Q

How does PAS work?

A

Periodic Acid or its sodium salt oxidizes glycols to aldehydes, which are made visible by exposure to Schiff’s Reagent

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13
Q

What is Schiff’ Reagent made from?

A

Basic Fuchsine and thionyl chloride

stains nuclei

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14
Q

What is the counterstain for PAS?

A

Fast Green FCF (stains cytoplasm and collagen)

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15
Q

What does PAS stain?

A

Carbohydrates (basement membrane, mucus)

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16
Q

Congo Red

A

Direct cotton dye

hydrogen binding to carbohydrates

elongated hydrophobic structure will bind amyloid fibrils under specific conditions (High pH, rinse with ethanol)

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17
Q

How does Congo Red stain work?

A

Hydrogen Binding to carbohydrates

Elongated hydrophobic structure will bind amyloid fibrils under specific conditions

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18
Q

What does Congo Red stain?

A

Amyloid deposits (shown in polarized light (green))

protein carbohydrates

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19
Q

What is a unique feature of Congo Red Stain?

A

unique feature of amyloid binding: dichroism

Reddish staining in bright-field

Bright Apple Green birefringence under polarized light (glows) → shows amyloid deposits

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20
Q

Where is Congo Red Staining mostly used?

A

Pathology

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21
Q

Alcian Blue

A

stains acid mucosubstances

does not usually stain nuclei or RNA

use pink or red counterstain (PAS is compatible)

different pHs stain dif things

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22
Q

What does Alcian Blue stain?

A

acid mucosubstances

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23
Q

What does Alcian Blue usually NOT stain?

A

Nuclei or RNA

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24
Q

What happens to a cell with Alcian Blue staining at pH = 2.5?`

A

all acid mucosubstances stain

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25
What happens to a cell with Alcian Blue staining at pH = 1?`
only sulfated acid mucosubstances will stain
26
What is a counterstain for Alcian Blue?
pink or red counterstain (PAS is compatible)
27
What is a counterstain for PAS?
Fast Green FCF
28
Masson's Trichrome
stain nuclei with iron-hematoxylin (brownish black) stains cells and connective tissue different colors Counterstain with Fast Green FCF (cytoplasm and CT) or aniline blue and Biebrich Scarlet-Acid Fuchsine (Type I Collagen) must be careful with fixative used
29
What does Masson's Trichrome stain? with what?
Nuclei → Iron-hematoxylin Cytoplasm and CT → Fast Green FCF or aniline Blue Type 1 Collagen (Muscle, Cytoplasm, Keratin) → Biebrich Scarlet-Acid Fuchsine
30
What fixatives work well with Masson's Trichrome?
Mercury-containing like SUSA or Zinc-formaline (Kiernan)
31
What is used as a counterstain for Masson's Trichrome?
Fast Green FCF (cytoplasm and CT) or aniline Blue and Biebrich Scarlet Acid Fuchsine (Type I Collagen)
32
Which counterstain is the differentiation step in Masson's Trichrome?
Biebrich Scarlet-Acid Fuchsine it stains muscle, cytoplasm, and keratin red
33
Prussian Blue
Reacts chemically to iron present in the tissue and produces an insoluble blue pigment (ferric ferrocyanide) detects cysteine residues, serotonin, catecholamines, and melanin precursors
34
What is Prussian Blue made of?
Ferric Ion and ferricyanide
35
What does Prussian Blue Detect?
detects cysteine residues, serotonin, catecholamines, and melanin precursors
36
What is Prussian Blue used for pathologically?
to detect iron deposits in tissue
37
Mucicarmine
Stains Epithelial mucins deep red Carmine stains nuclei adds aluminum, changing chemistry will also stain cryptococcus neoformans (fungus)
38
What does Mucicarmine stain? what color?
epithelial mucins → deep red carmine stains nuclei fungus (Cryptococcus Neoformans)
39
What is Carmine from Mucicarmine made from?
ground insects or synthetic alternatives
40
Verhoeff-van Gieson (VVG)
* Stains elastic fibers * Two parts: * Verhoeff's stain →stains elastic fibers nuclei, and myelin sheaths black * Van Gieson's reagent → stains collagen red (Acid fuchsine) and cytoplasm yellow (Picric Acid)
41
What does VVG stain? what color?
elastic fibers, nuclei, myelin sheaths → black collagen → red cytoplasm → yellow
42
What is VVG made of?
* Verhoeff's stain → iron hematoxylin + iodine, potassium Iodide * Van Gieson's reagent * Acid Fuchsine (red) * Picric acid (yellow)
43
Acid Fast-Bacilli (AFB)
Stains red for mycobacteria (includes causative agents for TB and leprosy) Ziehl-Neelson method Uses heat (and sometimes phenol) to permeabilize bacterial cell walls: dye retained after cooling uses basic fuchsine or pararosaniline counterstain with methylene blue
44
What does AFB stain? what color?
Mycobacterium (including causative agents for TB Leprosy) → Red
45
How does AFB work?
Uses heat (and sometimes phenol) to permeabilize bacterial cell walls dye retained after cooling Uses Basic Fuchsine or Pararosaniline
46
What do you counterstain AFB with?
methylene blue
47
Gram Stain
* Stain with Crystal Violet (checks cell proliferation) * precipitate dye with iodine-potassium iodine solution * extract insoluble dye with organic solvent (etOH, Acetone, aniline) * gram negative → don't retain dye * gram positive → retain dye, it cannot be extracted * Stain with safranin * stains gram-negative red
48
Gram-negative
Bacteria that lose dye stained red with safranin
49
Gram positive
bacteria that retain dye in a gram stain fungal hyphae and spores are gram positive
50
Gomori Methenamine Silver Nitrate (GMS)
* Detects bacteria known as spirochetes * carbohydrates in fungal wall * stains fungi, cellulose, starch, and chitin black * also stains melanin granules, glycogen, or mucus * counterstain: Fast Green FCF
51
What does GMS use?
Chromium trioxide, sodium metabisulfite, methenamine-silver stock (with borax for working solution)
52
What was Gomori looking for when using GMS?
glycogens and mucus
53
What does Gomori Methenamine Silver Nitrate (GMS) stain? what color?
Fungi, cellulose, starch, and chitin stains black also stains melanin granules, glycogen, or mucus
54
What is the counterstain for Gomori Methenamine Silver Nitrate (GMS) stain?
Fast Green FCF
55
Reticulin
* Used for reticular fibers and basement membrane * collagen fibers with hexose sugars bound * similar to PAS but uses silver * can use trichrome (Lillie's allochrome) * Fibers are birefringent using picro-sirius red * but basement membranes will not show as well
56
What does Reticulin stain for?
Reticular fibers and basement membranes
57
What can be used to counterstain Reticulin?
* trichrome (Lillie's Allochrome) * Picro-sirius Red (stains fibers red) * basement membranes won't show well
58
What are the acidic (acidophilic) dyes?
* Eosin * Picric Acid (Verhoeff-Van Giesen VVG) * Acid Fuchsine (collagen) * Fast Green FCF (CT) * Biebrich Scarlet * Picric-Sirius red
59
What are the basic (Basophilic) dyes?
* Hematoxylin * Methylene Blue (Giemsa) * Basic Fuchsine (pararosaniline) * Crystal Violet (Gram Stain) * Cresyl Violet (Nissl Stain) * Orcein (mixture; elastin) * Safranine
60
What is osmium stain used for?
lipids myelin sheath around nerves fixes lipids in place
61
What is Periodic Acid Schiff (Pas) used for?
Carbohydrates (glycogen in hepatocytes)
62
What is silver stain used for?
proteins nerve fibers, reticular fibers
63
What are trichrome stains used for?
will stain connective tissue components different colors
64
What happens when lipids/carbs are lost during fixation?
Dark purple areas show protein only lighter color areas are heavily glycoylated
65
Why is osmium better than H&E for staining the myelin sheath?
* H&E * can't really see myelin sheath * Osmium * stains myelin sheath black * can be used to see damage to myelin sheath
66
What stain is better for Nerve fibers? H&E or Silver?
Silver; makes it easier to see nerve fibers
67
What do you do if you cannot distinguish between cells morphologically?
* Look at the proteins they express * B cells vs. T cells * NK cells vs. T Helper cells * Glial Cells in the CNS * they all looks similar but express different proteins
68
How do you histologically look at protein expression?
* Immunohistochemistry (IHC) * on cultured cells → immunocytochemistry (ICC)
69
How does immunohistochemistry work?
It uses antibodies to recognize specific proteins it detects antibodies through bound moieties (direct or indirect)
70
What is a direct moiety in IHC?
moiety bound to a primary antibody
71
What is an indirect moiety in IHC?
moiety bound to secondary antibody that recognized primary antibody
72
How does IHC work?
1. Fluorescein isothicynate (FITC) 2. Antibody that recognizes antibody (ed. Goat anti-rabbit) 3. Antibody that recognizes protein of interest (e.g. Akt) 4. Cell growing on glass surface (or cell in tissue slice on glass slide) 1. fixed and permeabilized
73
How is IHC visualized?
Colorimetric → Visible on light microscope Fluorescent → more sensitive
74
What is an issue with Non-IHC H&E Staining of the Epidermis?
Can see Keratinocytes but not Langerhans cells
75
What is the pro of colorimetric IHC staining?
You can see Keratinocytes (purple) and Langerhans Cells (Gold)
76
What is the pro oof Fluorescent IHC Staining?
Good for showing the morphology of Langerhan Cells but Cannot see Keratinocytes
77
How are fluorescent dyes classified?
* based on excitation/emission spectra * light absorbed at one wavelength (excitation * light emitted at lower energy (higher wavelength)
78
What are commonly used fluorophores? What color do they show?
DAPI → blue FITC → Green Cy3 → Red
79
How do you determine which stain to use?
Based on what structure you want to look at and the chemistry of the stain
80
What are the major organelles and their general functions in mammalian cells?
* Nucleus → information storage * Cytoskeleton → structure * Endoplasmic Reticulum → protein/lipid production * Mitochondria → Energy production * Golgi Apparatus → Protein production * Cytoplasm → Ionic balance, protein aggregates
81
Define: DNA
higher order organization by histones
82
Define: Nucleolus
ribosome production proteins imported from cytoplasm
83
Define: Euchromatin (EC)
unraveled chromatid Doesn't stain as intensely b/c it is unraveled actively transcribed
84
Define: Heterochromatin (HC)
Condensed chromatid not actively transcribed The more HC the more inactive
85
What does a flat/compacted nucleus mean?
it is inactive
86
What does a round nucleus mean?
it is active
87
Define: Microfilaments
Structural (red)
88
Define: Microtubules
Organization (green)
89
Define: Intermediate Filaments
signal transduction
90
Define: Endoplasmic Reticulum
Protein (rough) and lipid (smooth) production Specific conditions required for normal protein folding calcium storage
91
Define: Mitochondria
Energy production by protein complexes across inner and outer membranes Matrix has specific chemistry (highly eosinophilic-pink areas) Calcium Storage Closely associated with microtubules and ER
92
Which star has more mitochondria?
* Red star * more eosinic * more energy * more active transport * more mitochondria (pinker) * alot of mitochondria = stain heavily with Eosin
93
Define: Golgi Apparatus
Found to one side of the. nucleus Vesicle production Protein processing and sorting Associated with microtubule organizing center (MTOC) doesn't typically stain
94
What is an artifact?
Debris or damage from preparation of the tissue
95
What are the types of artifacts?
Separation Tears Folds Dust Bubbles Chatter Loss of delicate structures
96
What is a Separation artifact?
Most common between two types of tissues tissue separates organelles pull apart
97
What is a Tear artifact?
Found in the middle of tissue tissue rips during preparation nothing lining it (lumen is usually lined with epithelia)
98
What is a fold artifact?
Tissue folds during preparation Common in cartilage
99
What is a Dust artifact?
Dust particles get trapped in slide during preparation
100
What is a chatter artifact?
Blade used to cut tissue doesn't cut through tissue smoothly changing blade helps avoid causes lines in tissue
101
What is a loss of delicate tissues?
Area cibrosa in the renal pyramid tissue gets removed during preparation
102
What is photomicrography?
Taking pictures of cells
103
What are the two types of cameras?
Non-digital (rare) Digital
104
What is a non-digital camera?
Best quality on color reversal film (slides) requires a projector images must be scanned for digital (What Neary Used for Thesis)
105
What is a digital camera?
* Basic will capture color * More advanced will capture pixels * pseudo-coloring used for fluorescent channels * Best files are high resolution TIFF (can use JPEG)

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