Histology- Exam #1

What is histology?

The study of tissue

What is a tissue?

a group of cells that share the same function

What is histopathology?

the study of tissue failure

(histo= tissue and pathos= suffering)

Who is Marcello Malpighi?

• lived 1628-1694

• italian doctor

• father of histology

• first to discover capillaries

Who was Marie Francois Xavier Bichat?

• lived 1771-1802

• french anatomist

• recognized that organs were composed of multiple tissues

• first to name tissues

What are skills that make a good histologist?

• observant/attention to detail

• organized (multitasking)

• strong background in (bio)chemistry

• teamwork

• patience

What stain was used for these slides?

H&E (hematoxylin & eosin)

What are the different histology jobs?

• clinical/research settings

  • hospitals

  • universities

  • veterinary hospitals

• state and federal agencies

  • e.g. CDC, department of health

• professor

• private industry- R&D, contract work

• pathologists (MD)

What is parental investment?

provisioning of resources to offspring at expense of future offspring

What is Calcium important for?

• intracellular communication

  • nerve transmission

  • muscle contraction

• hemostasis

• pH buffer

• structural component of bone

What are the types of microscopy?

• light (bright-field microscopy)

• polarizing

• phase-contrast

• dark-field

• fluorescence

• confocal

• electron

• atomic force

What kinds of microscopy can be used with unstained cells?

• phase-contrast

• dark-field

Describe light (bright-field) microscopy.

• reflective or transmitting

• limited to dark or strongly contrasting objects

• max resolution is 0.2 μm

Describe polarizing microscopy.

• extension of bright-field microscopy but with filters

• looking for high ordered objects

• can be used to detect silicosis

What kind of microscopy should be used to detect silicosis?

polarizing microscopy

What is amyloidosis and what mode of microscopy is used to detect it?

• amyloidosis: abnormal deposition of amyloid proteins in tissue and organs

• amyloids are highly-ordered with lots of beta sheets

• examples of diseases associated with amyloidosis: alzheimers, type 2 diabetes, parkinsons, atherosclerosis, TSE (prion based disease)

• amyloids stained with congo red

• detected with polarizing microscopy

Describe phase-contrast microscopy.

• makes dense areas more bright

• reveals many structures without need for staining

• thus can be performed on unstained (i.e. living) cells

• can be used for cell culture research

• appear as bright and dark areas

Describe darkfield microscopy.

• also used with unstained cells

• light directed at specimen from indirect angles. only scattered light reaches objective lens

• field of view appears black

Describe fluorescence microscopy.

• not usually used for naturally fluorescent molecules

• used in cell biology/ histology to:

  • establish the intracellular distribution of a protein

  • characterize the dynamics of the protein distribution

  • suggest potential functions of the protein

• immunofluorescence techniques

• 
  

What is fluorochrome?

dye that fluoresces when excited by UV

What is the disease we discussed in class that can be detected using immunofluorescence techniques?

• IgA nephropathy (Berger’s Disease)

  • IgA is most commonly found in mucous membranes

  • nephropathy = pathology of the kidney

  • most common form of adult glomerulonephritis (inflammation of the glomerulus)

    • IgA clumps together and causes backup in glomerulus

What are the types of immunistaining?

Describe confocal microscopy.

• microscope that increases contrast and resolution by eliminating out of focus light

  • eliminates out of focus light by only allowing one plane to be visible

• give a better “3-D” perspective

• in focus = in focal plane

Describe electron microscopy and the two types.

• use electrons instead of light

• highest resolving power

• limitations: skill set

• types:

  • transmission electron microscopy: 2D, allows you to see organelles

  • scanning electron microscopy: 3D images

What are the steps of tissue slide production?

1. tissue fixation

2. tissue processing

   1. dehydration

   2. clearing

   3. embedding

3. sectioning

4. mounting

5. staining

6. coverslipping

What is tissue fixation and it’s functions?

• fixation = tissue preservation, ideally keeps cell in position it was in when you obtained the sample

• function:

  • 1. kills tissue to resist:

    • putrefaction: bacterial attack

    • autolysis

  • 2. maintain proper relationship between cells and extracellular components (keeps cell from getting smushed)

  • 3. aids in contrast among cell components

  • 4. aids in staining process

How does tissue fixation work? How are fixatives classified?

• actions- protein stabilization

  • renders enzymes inactive

• primary method is use of chemicals

  • classified as:

    • 1. additive or non-additive fixative

    • and as:

    • 2. coagulant or non-coagulant fixative

What is the difference between an additive and a non-additive fixative?

• additive fixative: a chemical that chemically binds to protein structure—link

• non-additive fixative: doesn’t bind or link; all non-additives must be coagulants

What are some common fixatives?

non-additives:

• alcohols

• acetone

• acetic acid

• these are very fast, but harsh on tissues (causes shrinkage and makes them brittle)

non-coagulants:

• formaldehyde

• glutaraldehyde (good for electron microscopy)

coagulants:

• picric acid (trichrome stains)

• alcohols

• acetone

What is one of the best fixatives?

• 10% NBF (neutral buffer formalin)

What are some factors affecting fixation quality?

• temperature

• size of tissue (easier to fix through small diameter)

• volume ratio (should be 10-20x more fixative than tissue)

• time

  • since death

  • duration of fixation

    • underfix= staining and decay problems

    • overfix= hardening

• choice of fixative

• penetration- goes along with size of tissue

• storage

• pH

  • neutral, most fixatives are buffered

• osmolality= staining of fixative

What are the steps of tissue processing?

1. dehydration

2. clearing

3. infiltration

4. embedding

Describe the dehydration step of tissue processing.

• process of removing water from tissue to allow for infiltration of embedding media

• embedding media, is water insoluble and thus won’t infiltrate hydrated tissues

What are some common dehydrating reagents?

• alcohols

  • ethanol (the best one)

  • methanol

  • isopropanol

  • butanol

• acetone

• universal solvents- dehydrate and clear (the next step)- faster

  • dioxan

  • tertiary butanol

  • tetrahydrofurane

What is the best dehydrating agent? How is it used?

• ethanol

  • fast procedure

    • ethanol (95%)

    • ethanol (absolute)

      • problems- alcohol > 70% precipitates phosphate salts making sectioning difficult

  • better procedure

    • ethanol (60-65%)

    • ethanol (95%) 2X

      • salt precipitation

    • ethanol (absolute) 2X

      • must be replaced at certain time intervals

Describe the clearing step of tissue processing.

• clearing: renders tissues transparent

• removal of dehydrating alcohols to allow for proper infiltration of embedding media

• commonly used:

  • xylene- best, aromatic hydrocarbon, quickly dissolves fats

  • toluene

  • benzene

  • chloroform

  • acetone

What is the best clearing agent?

xylene, but its unsafe

Describe the infiltration step of tissue processing.

• process by which clearing agent is replaced by embedding medium

• most common embedding media:

  • paraffin- needs to be melted; wanna go 2-3 degrees C above melting point → any hotter will harden tissue

  • water-soluble waxes

  • celloidin

  • plastics- epoxy resins, glycol methacrylate

• process:

  • paraffin heated a few degrees above melting point

  • cleared tissue is immersed often under a vacuum

Describe the embedding step of tissue processing.

• process of placing the infiltrated tissue into a block of the embedding medium

• proper orientation is key!

  • when cutting long things, need to orient at oblique angle

Describe the sectioning step of tissue processing.

• microtome

  • rotary

  • sliding

• cryostat

  • frozen

  • widely used in clinical setting

  • used when sample is stained with antibody

  • quicker and preserves protein structure better to allow antibody staining

  • cell quality is lower though

• tissue ribbons

  • hard to achieve

What is Mohs surgery?

• multi-step procedure to remove cancer without removing non-cancerous tissue

• fast, but downside is poor cell quality

• routine sectioning method yields better cell quality

Describe the mounting step of tissue processing.

• slides are coated with an adhesive to prevent tissue from falling off during subsequent staining

  • poly-L-lysine

  • elmer’s glue

  • albumin

  • these three (above) are additives for water bater

  • OR

  • (+) charged slides

• tissue is placed in a floatation bath

  • temp is a few degrees below melting point

    • when too hot, causes cracking

Xylene is most often used as a(n)

clearing agent

Describe the staining step of tissue processing. What primary stains are used?

• goal of staining is to increase contrast of cellular structures

• primary stains: hematoxylin & eosin (H&E)

  • eosin is an acidic dye

    • carries a net (-) charge so binds (+) charged molecules

    • pink color

  • hematoxylin mimics basic dye

    • binds to (-) charged particles (such as the phosphate backbone of DNA)

    • counterstain

    • blue color

    • nuclear stain

    • net (+) charge

    • always used in conjunction with a mordent (metal/salt)

    • stain some proteins; have (-) charged carboxyl group

      • stains some sulfate groups on glycosaminoglycans (sugar protein polymer)

    • because it’s a basic dye, anything it stains is “basophilic”= loving basic dyes

      • ex: basophils

      • stains a deep purple

  • eosinophilia / acidophilia = binds well to eosin/acidic dyes

    • stains red

    • ex: eosinophils

What is the haematoxylon campechianum?

• logwood tree

• endemic to central america

• cultivated in Jamaica since 1715

• hematein- is oxidized form

• used in pap smears

What is a sample H&E procedure?

• look at slides

• essentially dewaxing and hydrating your tissue

• “blueing” = make tissue look blue by toning down purple color

  • blue jean blue

• 1. dewax

• 2. hydrate

• 3. blueing

• 4. dehydrate

• 5. coverslip

What is progressive vs. regressive staining techniques?

• progressive: requires more checking to achieve the perfect staining

• regressive: overstaining, requires blueing to achieve the perfect stain

Describe Periodic acid-Schiff stain (PAS).

• specialized stain

• stains basement membrane, sugars & mucins

Describe trichrome stains.

• specialized stains

• Gomori, Mallory, Masson’s

  • useful for collagen staining → vivid blue; distinguish muscle & connective tissue

Describe Silver stain.

• specialized stain

• used for staining neurons

• stains black

What is Wrights stain used for?

blood

Describe Orcein stain.

• specialized stain

• ver hoeff

• for elastic tissue

• stains black

Describe the last step of tissue slide processing.

• coverslipping

• permount

  • allows for higher quality of slide

T or F: Positively charged molecules are readily stained by basic dies

false

Biomolecules/cell structures that are stained by acidic dyes are called __?

eosinophilic / acidophilic

What is an example of cell specialization?

• example: skeletal muscle

  • makes a tremendous amount of actin because it’s needed for muscle movement

What are the levels of organization?

What is the cytoplasm and it’s components?

• cytoplasm: non-nuclear portion of the cell

• includes:

  • organelles: little organs

  • cytoplasmic matrix: mostly water, has ions and organic metabolites

What are the membrane-bound organelles in the cell?

• plasma membrane

• endosomes

• lysosomes

• transport vesicles

• rough ER

• smooth ER

• golgi apparatus

• mitochondria

• peroxisomes

Describe the plasma membrane of the cell.

• sets boundaries

• fluid mosaic

• phospholipid bilayer

• compartmentalizes the cell

• allows things to be isolated/stored

• could have microvilli attached

• has peripheral proteins that provides linkage to cytoskeleton

How are endosomes, lysosomes, and transport vesicles formed?

all formed from invagination of cell membrane

Endocytosis and exocytosis need to be __ over time!

balanced

What is an endosome?

membrane bound vesicle formed as a result of endocytosis

• protein sorting

What is a lysosome?

• forms as a result of maturing endosomes accumulating acid

  • recycling center

  • have enzymes that degrade organic molecules

  • ruptured = causes autolysis

Describe exocytosis.

• exocrine glands

• pepsinogen is one of the enzymes released

• when looking at exocrine glands, they display eosinophilia because they are waiting to release their contents

Describe rough endoplasmic reticulum.

• has ribosomes on its surface

  • mRNA translation

• for protein synthesis

• basophilic

• ex: nissl bodies on a neuron, displays heavy protein synthesis

Describe smooth endoplasmic reticulum.

• for lipid synthesis

• steroid metabolism

• glycogen metabolism

• no ribosomes on surface

Describe the golgi appratus.

• “cell warehouse”

• protein modification

• doesn’t stain well

What are peroxisomes?

• for oxidation → detoxification in cells (kidney, liver)

  • degrade fatty acids

Describe the mitochondria.

• can use janus green to stain

• metabolism

• cell that has high metabolic demands have more numerous mitochondria

What are the membrane-free organelles in the cytoplasm?

• cytoskeletal elements

  • microtubules

  • actin

  • intermediate filaments

  • centrioles

• ribosomes

Describe actin.

• two forms:

  • fibrous

  • globular

• for muscle contractions

Describe microtubules.

• larger in size, quickly assemble and disassemble

  • important for mitosis and meiosis

  • cilia beating

  • highways in cells to move transport vesicles

  • associated with flagella and sperm

What are centrioles?

microtubule organizing center

What are intermediate filaments?

structural role in cells, no movement

What are the components of the nucleus?

• chromatin

• nucleolus

• nuclear envelope

• nucleoplasm

What is chromatin? What are the forms?

• DNA-protein complex

• two forms:

  • euchromatin: dispersed, unfolded DNA

    • unzipped DNA used for replication and transcription

  • heterochromatin: dense, folded

Describe the nucleus.

• membrane-bound organelle

• houses DNA (an extremely long molecule)

• DNA packed tightly around protein

How can you identify plasma cells?

have a clock-like pattern around nucleus (dot in center, ring around edge because of chromatin)

What are the 4 tissue classes?

1. epithelium- boundary between outside and inside world; is protecting something

2. connective tissue- link tissues together

3. muscle tissue- movement

4. nerve tissue- specialized, long term communication

Where are the general places you will find epithelium tissue?

1. surface of body

2. lines internal cavities (with the exception of joints)

3. tubes that communicate with outside world

4. glands/ducts

What are the functions of epithelial tissue?

1. secretion (ex: mucus)

2. absorption (ex: sugar, nutrients)

3. transportation (ex: cilia)

4. protection (ex: skin)

5. communication/ receptor function (ex: taste buds)

What are the characteristics of epithelial tissue?

1. free surface

2. avascular: no direct blood supply

3. basement membrane

4. morphological polarity: unique functions in different parts of the cell

5. tightly bound cells

What is cell polarity? What are the regions?

• unique cellular function in different parts of the cell

• regions/domains:

  • apical

  • lateral

  • basal

Describe the apical domain of epithelial cells.

• vary among epithelium

  • can have cilia, microvilli, etc.

• if have microvilli = increased surface area

  • efficient transport

  • all microvilli filled with actin filaments

  • pumps / transporters, enzymatic roles

• variability in size and density of microvilli

• cilia = larger projections than microvilli, microtubules found in cilia provide ability to move entire structure

• trachea = pseudostratified

What is Immotile Cilia Syndrome (ICS)?

• affects cilia of certain epithelial tissue

• abnormal microtubules

  • dynein arm connects microtubules

  • affects ~1/ 20,000 individuals

• common symptoms:

  • infertility (both genders but mostly men because it affects sperm swimming)

  • swelling of ventricles in the brain

  • respiratory distress = kartageners syndrome

• know picture!!

Describe the lateral domain of epithelial cells.

• adjacent cells linked to tissue

• CAM = cell adhesion molecule

• tight junctions: controlling/barriers to paracellular passage

Describe the junctions on the lateral domain of epithelial cells.

• tight junctions

  • link to cytoskeleton by actin filaments

  • controlling/barriers to paracellular passage

• anchoring junctions = desmosomes

  • maintain tissue integrity

  • structural role

  • have intermediate filaments

• gap junctions

  • cell-cell communication

  • ex: cardiac and smooth muscle cells (Ca²⁺)

Describe the basal domain of epithelial cells.

• epithelial links to underlying connective tissue

• PAS = periodic acid schiff

  • stains basement membrane and carbohydrates

How is epithelium classified?

• layers:

  • simple

  • stratified

• shape

  • squamous

  • cuboidal

  • columnar

• special

  • pseudostratified

  • transitional

• the structure dictates function!

Describe simple squamous epithelium, where its found, and its function.

• epithelium = layer of outside tissue

• found in blood vessels, kidney (Bowman’s capsule), alveoli in lungs, body cavity, capillaries

• on fragile tissues

• not a great barrier

• function: exchange because thin and one layer thick

What is atherosclerosis?

• atherosclerosis = formation of plaques under vessel

• buildup of cholesterol, immune cells, fibrotic tissue (= collagen), cellular waste

• plaques can break off and block vessels → heart attack

• vessels lined with simple squamous epithelium

• risk factors:

  • old age

  • obesity

  • smoking

  • diabetes

  • sedentary lifestyle

  • hypertension

  • hyperlipidemia = excessive LDL’s

Where is simple cuboidal epithelium found, and what is it’s function?

• found in kidney tubules, ovary, hormone glands, ducts of exocrine glands

• function: absorption or secretion

  • not really protective

  • mostly for transport

Where is simple columnar epithelium found, and what is it’s function?

• found in small intestine (digestive tract)

• function: absorption & secretion

• taller than they are wide

• one nuclei/ cell

• do a lot more transport/ secretion because of large cell size

• only one cell thick so easy to move things across without blockage

Where is stratified squamous epithelium found, and what is it’s function?

• wet areas = non-keratinized

  • vagina, esophagus, mouth

• dry = keratinized

  • skin

  • moisture loss prevention and protection

• function: protection

• cells at top definitely doesn’t touch basement membrane, so = stratified

• divides from bottom up

Where is stratified cuboidal epithelium found, and what is it’s function?

• locations: found in sweat gland ducts

• function: barrier, conduit, secretion

• always only 2 layers thick

• difficult to find

Where is stratified columnar epithelium found, and what is it’s function?

• locations: largest ducts of exocrine glands

• rare, found in eye

• function: barrier, conduit

Where is pseduostratified epithelium found, and what is it’s function?

• found in trachea

• function: absorption, secretion, movement

• looks very stratified but isn’t because all cells are touching the basement membrane

• changes shape to fit more cells

• always ciliated

• cells don’t sit straight

Where is transitional epithelium found, and what is it’s function?

• found in urinary bladder, ureter, and urethra

• function: barrier and stretchability

  • done by layering of different cell sizes

    • top layers bigger than underneath (top cells = umbrella cells)

• stratified

Describe epithelioid tissue.

• “epithelium-like”

• lack a free surface

• usually associated with endocrine function

  • ex: Leydig cells of testis

• function: hormone (endocrine) production/release

• vascularized (has blood vessels)

• found in testes, ovaries, thyroid, pancreas (islets), pituitary