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Body Logistics :) > Histology > Flashcards

Histology Flashcards

1
Q

Tissue DEFINTION

A

A Latin word that means woven

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2
Q

4 broad classifications of tissues

A
  1. Epithelial;
    - polarised at surfaces
    - often on edge/ surrounding
  2. Connective
    - made of cells, extracellular proteins/glycoproteins and gels
    - main cells are fibroblasts, chondrocytes, osteoblasts/osteocytes/osteoclasts/ stem cells/ bone marrow/ adipocytes
    • main products are fibres, ground substance, wax and gel like materials
  3. Muscle
    • skeletal, cardiac, smooth
    • main function to contract (movement, stability, movement of tissue contents) also secretes hormones (Natriuretic factors, myostatins)
  4. Nerve
    • fast communication system, bundle to nerve fibres
      - nerves can be dissected and seen by the eye
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3
Q

Limit of resolution definition

A

The SMALLEST distance by which two objects can be separate and still distinguishable as 2 separate objects

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4
Q

6 comparisons of light and electron microscopy

A

Light can view in natural colours whilst electron is b&w
Light has a large field of view, electron is limited (100 um)
Cheap and easy prep for light, difficult and expensive prep for electrons
Light can view living and moving; electron has to be dead and inert
Light magnification is x600 whilst electron is x500,000
Light res is 0.25um, electron res is 0.25nm

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5
Q

Biggest and smallest cells visible to the human eye

A

Biggest is the ooctye

Smallest without nucleus is platelet, with nucleus is the spermatozoa

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6
Q

TEM vs SEM

A

TEM; fix with Glutaraldehyde, embedded in epoxy resin, stain, use microtome with diamond knives

SEM; Fix with Glutaraldehyde, embedded in Epoxy resin, stain

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7
Q

What are light microscopy, TEM and SEM used for????

A

Light - good for cells and tissues
SEM - surface of cells
TEM - intracellular structures and organelles bs osmium teroxide is used as a fixative which binds well to membranes

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8
Q

Gross anatomy

A

Use of senses to examine a sample, able to distinguish between objects 0.2mm apart

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9
Q

Considerations for histology

A
  • thin (2 to 20um)
    -translucent
    -needs to fit equipment
    -fixation (preservation of tissue) to prevent putrefaction (rotting)
    (Note: fixation isn’t necessary if being used in cell culture or as a diagnostic frozen section)
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10
Q

Process for light microscopy

A
  1. Fixation - preserve sample in formalin (37% aqueous solution of formaldehyde with 0.9% NaCl solution -saline)
  2. Embed the tissue in a substance allowing it to be sliced thinly (5um) MELTED PARAFFIN WAX is used as it sets hard when cool
  3. Stain to see components - H&E
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11
Q

What type of formalin is used in fixation and how long is sample left in formalin for

A

Isotonic with the intracellular fluid to allow better penetration of formaldehyde

This reacts with amino groups in amino acids forming a methylated bridge between protein chains under RAPID FIXATION.

Left for 24-48 hours. Any longer results in dehydration and thus tissue shrinkage causing fixation artefacts

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12
Q

Methods to obtain biological specimen

A
  1. Surgery and dissection
  2. Scraping methods
  3. Sharp needles (needle biopsy, endometrial biopsy)
  4. Direct venepuncture
  5. Pipeline
  6. Hysterectomy
  7. Occassionally body fluids are examined
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13
Q

After fixing, paraffin is used in embedding. What happens in the process leading up to embedding

A

Specimen washed
dehydrated in alcohol solutions of different concs
Then in solvents miscible with alcohol and hot paraffin wax
Paraffin wax added and left overnight before more paraffin wax added

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14
Q

What happens after the block of tissue is cooled and hardened?

A
  • Mounted on a microtome
  • Thin sections cut with -sharp steel blade -sharp edged glass -diamond knife
  • fragile sections moved with paintbrush to warm water bath. The surface tension causes sample to stretch to reduce artefacts.
  • then to slide with sticky substance (albumin, silica) to allow it to adhere closely to slide when dry
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15
Q

Why must any sample be stained and how can this be done

A

Paraffin is colourless

  1. Paraffin dissolved and rehydrated with alcohols
  2. H&E can be used where tissues are immersed in aqueous haematoxylin, washed, transferred to alcohol (eosin more soluble in alcohol than water), immersed in eosin
  3. Wash again, transfer to non aqueous mounting medium, add coverslip
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16
Q

Which methods of tissue processing can be used to observe neutral fats and which cannot

A

Frozen sections and dyes soluble in fats need to be used
To retain membrane structures, heavy metal fixatives eg permanganate and osmium are required.

Xylene and toluene solvents aren’t used as sugars aren’t detectable and lipohillic molecules are stripped out of the tissue

17
Q

What can be seen when using H&E and why

A

Haematoxylin is a BASIC dye and thus binds to acidic DNA and RNA. Nucleus stained BLUE more strongly

Eosin is an ACIDIC dye and thus binds to basic intra/extracellular proteins, cytoplasm, collagen, elastic fibres etc) Cytoplasm and extracellular matrix stained PINK most strongly.

18
Q

Why may a Frozen section be used?

A

Compared to paraffin wax formalin fixed frozen sections….

  • use fresh tissues instead of fixed (adv and disadv)
  • quick 10-20 mins
  • yet saving time only months not permenant
  • uses intraoperative consultation instead of pathological diagnosis
19
Q

Outline of frozen section

A

Specimen on metal disc frozen quick to -20 to -30 degrees
Specimen cut with microtome is cryostat in a freezer
Stained with H&E

NOTE QUALITY OF SECTIONS IS NOT SO GOOD.

20
Q

Immunohistochemistry and immunofluorescence?

A

BOTH use ANTIBODIES that target ANTIGENS which are 5 to 7 amino acids long, due to complementary shape.

Immunoflorescence; fluorescent dyes visible under UV light eg in CONFOCAL MICROSCOPY

Indirect immunohistochemistry; use of enzyme activated secondary antibody complexes that are coloured in interaction

21
Q

What enzymes are used in indirect immunohistochemistry and why may it be used??

A

Peroxidases (horse radish peroxidase is absent from human tissue)
BROWN PPT; peroxidases, 3,3-diaminobenzindine and peroxide
COLOURLESS PPT; acid phosphatases, glycerophosphate and lead nitrate
BLACK PPT(of lead sulphide); ammonium sulphide

Therefore used to identify organelles containing acid phosphatase (lysosomes)

22
Q

What are antigen retrieval methods and when can/ cannot this be used in immunohistochemistry

A

The tissue processing procedures may alter target antigen structure. Therefore this (partial digestion of fixed proteins/ antigen retrieval) work on ROBUST tissue. NOT on tissue less robust like adipose so frozen section is needed

Tissue is heated with weak acids (citric, EDTA)

23
Q

How can 2D images be used to form 3D one

A
  • multiple images taken at 1-10um distance and stored on a computer
  • it lines them in order creating a Z stack
  • therefore can be seen in 3D x,y,z

Can be used in the evaluation of various eye diseases, quantification of endothelial cells of the cornea and retina

24
Q

How to prep live cells into a culture

A
  • cutting and dicing
  • collagenase and DNAase
  • centrifugation
  • growth medium
  • culture (phase contrast microscope)
25
Q

Adv/disadv of cell culture

A

Advantages:

  • control over the physical environment
  • homogeneity of sample
  • less need for animal models

Disadv:

  • hard to maintain (infection may occur)
  • small amount is grown at high cost
  • dedifferentiation
  • instability/aneuploidy
  • 3D architecture lost
  • influence of other cells/tissues not maintained
26
Q

Dark field

A
  • living cells
  • light is used that won’t be collected by the objective lens
  • dark background with light objects
  • also used in electron microscopy images

Body Logistics :) (5 decks)

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