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202: Histology I > Intro to Histological Techniques ✅ > Flashcards

Intro to Histological Techniques ✅ Flashcards

1
Q

Histology

A

Study of fine detail of biological cells and tissues

Uses microscopy to look at specimens of tissues prepared using histological techniques

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2
Q

Histology basic def

A

Microscopic study of normal tissues

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3
Q

Histopathology

A

Study of pathology in tissue

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4
Q

Pathology

A

Study and diagnosis through examination of surgically removed -organs,
-tissues (biopsy samples)
-bodily fluids
-sometimes- whole body (autopsy)

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5
Q

Biopsy

A

Procedure

To remove a piece of tissue or sample of cells from body
-so it can be analyzed in a lab

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6
Q

Tissue prep name

A

Paraffinization

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7
Q

Paraffinization steps

A
  1. Fixed
  2. Washed
  3. Dehydrated
    -in a series of alcohol solutions (increasing concentration)
  4. Cleared
    -xylol or toluol to remove alcohol
  5. Embed in melted paraffin
  6. Sectioned
  7. Stained

*steps 3-4 done with histokinette- automated step

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8
Q

Fixation

A

37% formaldehyde solution

-most common fixative
-tissues structure not altered
-generates weak H-bonds between proteins

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9
Q

Embedding

A

Solidified paraffin block trimmed to appropriate size & sectioned with slicing machine (microtome)

  1. dispense wax
  2. align tissue
  3. cool in place
  4. ID bead
  5. top-up wax

6.cool plate

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10
Q

Frozen tissue prep

A

For short term & faster use-> can rapidly freeze specimens
-instead of paraffin embedding (OCT compound in liquid N)

Cryostat used for cutting sections

Integrity isn’t great if frozen too fast
-the membranes break open

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11
Q

What can you do with tissues that are to be sectioned frozen?

A
  1. Frozen UNTREATED- in liquid N
  2. Sectioned
  3. Stained- mostly H&E staining
    -usually done for fast assay (in operating theatre)
  4. Fixed w mild fixative
  5. Cryo-protected in sucrose (10%, 20%, 30% sucrose)
  6. Embedded in OCT
  7. Frozen in -80 C
  8. Sectioned
  9. Stained- used for IHC or IF (these methods use specific antibodies against protein under investigation)

*IHC= immunohistochemistry
**IF= immunofluorescence

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12
Q

How thick are the tissue sections in:
1. Optical microscope

  1. EM?
A
  1. 5-10 microm
  2. 50-100 nm
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13
Q

Histochemistry

A

Histo + chemistry = chemistry in tissues

-one of most widely used techniques to help scientists localize and visualize cellular components, tissues and other living structures

-using diff stains and indicators (which react w the cellular components) to develop tiny colored structures that could be easily observed under a microscope

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14
Q

Acidic dyes in Histochem

A

Neg net charge on colored portion

React with + (catatonic groups) cell and tissues- particularly w amino groups of proteins

ACIDOPHILIC

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15
Q

Basic dyes in Histochem

A

Pos net charge on colour end portion

React w anionic components (-) of cell and tissues
-phosphate groups on nuclei acids
-sulphate groups of gylcosaminoglycans
-carboxy terminal of proteins

BASOPHILIC

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16
Q

Common dyes

A

Haematoxylin & Eosin

-most common

-mostly used to display structural features of the tissue

17
Q

Haematoxylin

A

Purple basic dye

Stains acidic macros
-DNA (nuclei),
-RNA (cytoplasm of cells with many ribosomes eg in glands),
-some proteins

18
Q
A

Pink acidic dye

Stains basic macros
-muscle cell proteins,
-collagen (a major extracellular protein),
-cytoplasm of most cells

19
Q

IHC

A

Visualization of distribution & localization of specific proteins in cells and in their proper histological context

Through indentification of antigens (proteins) using antibodies- that specifically bind to presented antigens

Types
1. Chromogenic IHC
2. Fluorescence IHC

20
Q

Chromogenic IHC

A

Specific binding of primary antibody

Secondary antibody conjugated to an enzyme eg HRPO

Addition of substrate eg DAB will generate a colored precip

-used to indetify specific molecules in the tissue eg proteins, usually overexpressed in pathologies

21
Q

Hybridization techniques

A

Specific sequences of RNA or DNA can be detected by hybridizing it with a fluorescently-labeled complimentary probe

Takes place in cell -in situ
Probes can be radioactively or fluorescently labeled

FISH- fluorescence in situ hybridization is used in genetic testing

22
Q

Light microscopy types

A

A. Normal light
i. Bright field
ii. Phase-contrast
iii. Dark Field

B. Ultraviolet light
i. Fluorescence

C. Laser beam and scanning
i. Confocal

23
Q

Electron microscopy types

A

A. Electron beam
i. Transmission Electron Microscope (TEM)
ii. Scanning Electron Microscope (SEM)

24
Q

Phase contrast microscopy

A

Enhance the contrast of images of transparent and colourless specimens

Allows the examination of unstained cells and tissues

–very useful for the examination of living cells

25
Q

Dark field microscopy

A

No direct light source
– objects appear bright on a dark background

Clinically used in:
– examining urine for crystals, such as those of uric acid and oxalate
– demonstrating specific bacteria such as spirochetes and the microorganism that causes syphilis

26
Q

Fluorescence microscopy

A

Detect naturally fluorescent molecules eg. Vitamin A

Immunodetection (fluorescent antibody)

Utilizes UV light

Immunofluorescence- usually confocal microscope is used

Qualitative analysis

27
Q

Confocal microscopy

A

Combines components of a light optical microscope with a scanning
system
-to dissect a specimen optically

Allows visualization of biologic specimen in 3D

28
Q

Which disciplines would use LM?

A

Histochemistry (tissue morphology)

Immunohistochemistry (quantitative analysis)

29
Q

EM

A

Electron beam used instead of light

Increases resolution x1000

30
Q

TEM

A

High res
-tissue structures must be well preserved

Tissues can be stained with heavy metals
-to detect specific structures

31
Q

SEM

A

Electron beam doesn’t pass through
-scans surface

32
Q

Sense of scale: LM vs EM

A

LM:
-Cells= ~25 microm (range ~6-100)
-Nuclei: ~10 microm (5-20)
-Red blood cell: ~8 microm

EM:
-Mitochondria: ~300 nm wide (200-400) x 3 microm (2-6)
-Ribosomes: ~15 nm
-Unit membrane (bilayer): ~10 nm across

202: Histology I (16 decks)

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