Ch. 1 - Histology & Its Methods of Study Flashcards Preview

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Flashcards in Ch. 1 - Histology & Its Methods of Study Deck (58):
1

DAPI and Hoechst stain

Specifically bind DNA
Used to stain cell nuclei
Emit a characteristic blue fluorescence under UV

2

Histology

The study of the tissues of the body and how these tissues are arranged to constitute organs

3

Fixation

Small pieces of tissue are placed in solutions of chemicals that preserve by cross-linking proteins and inactivating degradative enzymes

4

Dehydration

The tissue is transferred through an ascending series of alcohol solutions, ending in 100%, which removes all water

Water and paraffin aren't miscible

Can't go directly to 100% EtOH (tissue trauma)

5

Clearing

Alcohol is removed in toluene or other agents in which both alcohol and paraffin are miscible

Aka intermediate agent

Usually an organic compound; alters the refractive indicies of the tissue which makes it clear

Organic solvents dissolve lipids so must use cryotomy

6

Infiltration

The tissue is placed in an ascending series of melted paraffin until it becomes completely infiltrated

No higher than 58C or the tissue will denature

7

Embedding

The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden

Orientation of the tissue is important

8

Trimming or Sectioning

The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome

Makes serial sections ~ 5-7 microns thick

9

Microtome

Instrument used for sectioning paraffin-embedded tissues for light microscopy

10

Fixative

Stabilizing or cross-linking compounds used to fixate tissue samples
Different fixatives for different purposes

11

Basophilic cell components

Anionic
Stain with basic dyes
Ie: nucleic acids

12

Acidophilic cell components

Cationic
Stain with acidic dyes
Ie: proteins with many ionized groups

13

Basic dyes

Stain basophilic cell components
Toluidine blue, alcain blue, methylene blue, hematoxylin

14

H&E stain

Hematoxylin (basic dye, stains acidic material i.e. nucleus) and eosin (acidic dye, stains basic material i.e. cytoplasm)
Most commonly used staining method

15

Trichromatic dye

Mallory stain, Masson stain

16

Acridine orange

Fluorescent compound that binds to nucleic acids in DNA and RNA

Can distinguish between the two

Used in fluorescent microscopy

17

Fixative properties

Preserve tissue
Harden tissue
Inhibit bacterial growth (bacteria decompose tissue)
Block autolysis

18

Autolysis

Autolysis of the cell
Due to lysosome ruptures containing hydrolytic enzymes

19

What is the most common fixative?

10% buffered formalin
pH ~ 7.5

20

Cryotomy

Freezing the tissue before sectioning
Preserves the tissue structure

21

Staining

1. Clear paraffin
2. Rehydrate via descending series of alcohols
3. Stain with dye

22

Slide Prep

1. Obtain tissue sample and fixate
2. Dehydrate (ascending series of EtOH)
3. Clearing (of EtOH)
4. Infiltration (paraffin)
5. Embedding (paraffin block)
6. Sectioning (use microtome)
7. Staining
a. clear paraffin
b. rehydrate (descending series of alcohols)
c. stain
8. Dehydrate (to preserve dye)
9. Clearing (of dye)
10. Cover slip

23

Condenser

Collects and focuses a cone of light that illuminates the object

24

Objective lens

Enlarges and projects the image of the object to the eyepiece or ocular lens

25

Eyepiece or Ocular lens

Magnifies the image

26

Total magnification of light microscope

Objective X Ocular

27

Resolving power or Resolution

The smallest distance between two points at which they can be distinguished

28

What is the relationship between magnification and resolution?

Directly proportional

29

Fluorescence Microscopy

Tissue sections are irradiated with UV light, which appears brilliant on a dark background

30

Phase-Contrast Microscopy

Light Microscope

Can be used with living cell cultures

Parts of the cell appear lighter or darker depending on how fast light travels through them due to refractive indices

No fixation or staining

31

Differential Interference Microscopy (DIC)

Light Microscope

A modification of phase-contrast
Produces an image of living cells in 3D

32

Confocal Microscopy

Light Microscope

Uses a small point of light from a laser through a pinhole for better resolution and sharper focus than a bright-field microscope

33

Polarizing Microscopy

Light Microscope

Based on vibrations

Sample is placed between two polarizing filters and the axis of light rotates and the sample appears as a bright structure against a dark background

34

Birefringence

Ability to rotate the direction of vibration of polarized light

Feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin filaments

35

Bright-field microscopy

Light Microscope

Ordinary light passes through a thin section of the specimen from below, producing a 2-D image

36

Light microscopy

Microscopy that is based on the interaction of light with tissue components

Used to reveal and study tissue features

37

Electron microscopy

Microscopy based on the interaction of tissue components with beams of electrons

Better resolution that light microscopy due to electron beams shorter wavelength

38

Transmission Electron Microscopy (TEM)

Electron microscope

Permits resolution ~ 3 nm
Magnification of up to 40,000 X for isolated macromolecules or particles

An electron beam is pointed at the specimen, where some of the electrons pass through the specimen and some interact with it (absorbed or deflected)

Creates a 2-D black, white, and gray image depending on the interactions of electrons

Brighter where electrons passed readily, and darker (more electron dense) where electrons were absorbed or deflected

39

Scanning Electron Microscopy (SEM)

Electron microscope

The surface of the specimen is dried and coated with a layer of heavy metal (gold), through which electrons do not pass readily

Creates a black and white 3-D image by interacting with the metal and producing secondary electrons, which are captured by a detector

40

Autoradiography

A method of localizing newly synthesized macromolecules (DNA, RNA, protein, glycoproteins, and polysaccharides) in cells or tissue sections

41

Primary cell culture

Cells cultured from a tissue or organ

42

Cell line

Cell types isolated from tissue are maintained in vitro for long periods of time and become immortalized

43

Transformation

Certain changes that promote cell immortality

44

Enzyme histochemistry

Aka cytochemistry

Method for localizing cellular structures using a specific enzymatic activity present in those structures

Usually use unfixed or mildly fixed tissue sectioned on a cryostat to preserve the enzymes and avoid adverse effects of heat and organic solvents

45

Steps of cytochemistry

1. Tissue sections are immersed in substrate of enzyme to be localized

2. Enzyme is allowed to act on substrate

3. Section is put in contact with a marker compound that reacts with a product of the enzyme-substrate reaction

4. The product from the marker precipitates over the site of enzymes, allowing the region to be localized microscopically

The product from the marker must be insoluble and visible by light but having color or electron mic or by having e- density

46

Phosphatases

Enzyme that splits the bond between a phosphate group and phosphorylated molecules

Used to ID alkaline and acid phosphates

47

Dehydrogenases

Enzyme that removed H+ from one substrate and transfers them to another

Used to ID mitochondria

48

Peroxidase

Enzyme that promotes oxidation of substrates with the transfer of H+ to H2O2, forming H2O molecules

49

How can we visualize specific molecules?

Using a tagged compound or a macromolecule that binds specifically with the macromolecule of interest
Must be visible with light or e- microscope

50

The most commonly used labels are _______ compounds and _______.

fluorescent; metal particles

51

What are some molecules that interact specifically with other molecules?

1. Phalloidin
2. Protein A
3. Lectins

52

Phalloidin

Interacts strongly with actin
Tagged with fluorescent dyes
Commonly used to demonstrate actin filaments in cells

53

Protein A

Obtained from S. aureus
Binds to the Fc region of IG antibodies
Can be used to localize naturally occurring or applied ab bound to cell structures

54

Lectins

Proteins or glycoproteins derived mainly from plant seeds
Bind to carbohydrates with high affinity and specificity
Different lectins bind to specific sugars or sugar residues
Tagged with fluorescent dyes
Used to stain specific glycoproteins, proteoglycans, and glycolipids

55

Immunohistochemistry

Histologic method using labeled ab used to detect specific proteins or other molecules of interest in cells and tissues
Requires an ab against the protein or molecule of interest

56

How are ab of protein x of a certain animal species produced?

The isolated protein is injected into a different animal species
If the protein's amino acid sequence is different enough for this animal to recognize it as foreign, then it will produce ab against the protein

57

What are the steps to immunohistochemistry?

1. A tissue section/cells believed to contain the protein of interest is incubated in a solution with the labeled ab to that protein
2. Ab binds specifically to the protein
3. Protein location can then be seen with either light or electron microscope

58

Polyclonal antibodies

Different groups (clones) of lymphocytes in the injected animal that recognize different parts of wanted protein x