Download Methods in Cell Biology

Methods in Cell Biology
Medical Microanatomy 602
Edie C. Goldsmith, Ph.D.
[email protected]
Introduction
• The material in this power point is covered in Chapter
1 of your textbook. I would encourage you to read the
text, particularly sections that are covered here.
• While we will not specifically cover this material
during lecture, you will be expected to know this
information for the exam. I can address any questions
you may have during the lab period or by email.
• Notice that in the notes section below each slide I
have added information and explanations for the
images/text on that slide when necessary.
Tissue Preparation
• Fixation
– Preserve tissue structure
– Chemical
• Formaldehyde/glutaraldehyde - cross-linking amine groups
• Alcohol – denature proteins
• Heavy metals (osmium tetraoxide) – oxidation of proteins to
form cross-links; also react well with phospholipids
– Freezing
• Cryoprotectant (OCT, sucrose solution)
• Rapid low-temp freezing
• Lipids and activity stains (enzymes)
• Not all fixative works for all structures
– Formaldehyde - not good for lipids
• Don’t over fix
• Dehydration & Clearing
– Prepare tissue for embedding
– Removal of water can cause tissue shrinkage
• Infiltration
– Mix of clearing agent and embedding media
• Embed tissue
– Prevents tissue damage during sectioning
– Embedding agents - Paraffin, plastic resins,
acrylamide
• Section, mount and stain
• Samples are dynamic during processing
• Some things get retained
– Nucleic acid – protein complexes, cytoskeletal proteins,
ECM proteins, lipid-protein complexes
• Some things get lost
– Small, soluble molecules (ions, carbohydrates), glycogen,
proteoglycans
– Can use special fixation or staining methods
Tissue Staining
Basophilic
• Stain with basic dye
[dye+Cl-]
• Toluidine blue, methylene
blue, hematoxylin, alcian
blue
• Nucleic acids, some
cytoplasmic components
(rRNA and rER),
glycosaminoglycans and
acidic glycoproteins
Acidophilic
• Stain with acidic dye
[Na+dye-]
• Orange G, eosin, acid
fuschin
• Mitochondria, cytoplasm,
secretory granules, ECM
proteins
Dye color does not determine whether a dye is acidic or basic
(see Table 1.2 in your textbook)
Nuclei stained with
hematoxylin
• Hematoxylin & eosin
(H&E) is the most
common dye
combination you will
see this semester.
– Hematoxylin – basiclike dye which stains
negatively charged
molecules (blue)
• i.e. Nucleic acids
Material stained with eosin
– Eosin - acidic dye
which stains positively
charged molecules
(pink)
• Cytoplasm (proteins)
Metachromasia
• When certain dyes bind some cellular structures, their color
shifts
• This is due to the presence of a large number of polyatomic
anions (SO42- and PO43-)
– This leads to aggregation of the dye which changes is absorption
properties
– The polyatomic anions are common in ground substance (particularly
cartilage); heparin granules in mast cells; rER in plasma cells
• Toluidine blue is an example of a dye the exhibits
metachromasia. As it name would imply, this dye usually
stains blue. But when it is used to stain mast cells, the granules
in these cells contain lots of negatively charged molecules and
the dye’s color shifts to a purple-red appearance.
Other notable stains
• Periodic acid-Schiff
(PAS)
– Carbohydrates (glycogen,
mucin, basement
membrane)
– Periodic acid + sugar
aldehyde
– aldehyde + bleached basic
fuchsin (Schiff reagent)
magenta color
PAS positive
Staining (mucus)
Orcein
• Also known as resorcinfuchsin
• stains elastin found in
elastic fibers
• The individual fibers in
this image are elastic
fibers stained with
orcein
Aorta
• Trichrome stains
– Employ 3 dyes
– Masson Trichrome– most
common
– Usually used to look at
collagen in the
extracellular matrix
• Nuclei (basophilic) –
blue/black
• Collagen – green or blue
• Cytoplasm, muscle, keratin,
erythrocytes – red
erythrocytes
nucleus
collagen
• Azan
– Stains collagen, basement
membrane, mucin
nucleus
• Nuclei – red
• Collagen - blue collagen
nephron
Picrosirius Red
• Collagen specific stain
• Observed under
polarized light the
collagen appears
yellowish-orange in
color.
• Observed under normal
bright field microscopy,
the collagen would have
a bright pink
appearance.
In this image of skeletal muscle, the collagen has
a bright, glowing orange appearance. The striated
structures are muscle cells.
Nissl
• This uses a basic dye
on neuronal tissue.
• Stains the rER in
neurons
– Referred to as Nissl
substance when
observed in clumps
• Can also be done
with methylene blue
N
Gold and Silver Stains
Renal cortex
Gold staining of motor end plate
in muscle
Silver stain of reticular fibers
(type III collagen)
String-like appearance in this image
Lipids
• Preserved in frozen
sections
• Stains used in light
microscopy
– Sudan black, Oil red O,
Sudan IV, Osmium
tetraoxide
• Common stain used for
electron microscopy
– Osmium tetraoxide
Black circles in this image are lipid
droplets in brown fat cells
Enzyme Histochemistry
• Localize enzymes within
tissue
• Don’t over fix (frozen
sections)
• Precipitated product
• Indirect method
• Variety of enzymes
– Acid and alkaline
phosphatases,
dehydrogenases, peroxidases
• Light and EM
Alkaline Phosphatase in rat kidney
(Gomori stain with a lead precipitate)
Immunohistochemistry (IHC)
• Rely on the use of
antibody directed
against molecule of
interest
– Usually protein
• Direct vs indirect
• Amenable to many
forms of detection
– Fluorescence
– EM (gold)
– Light microscopy (color
precipitate)
Blue material in this fluorescent image is a
cell surface protein that binds collagen.
a-amylase ab stained with Protein A - gold
Orange indicates sites of peroxidase reaction
(lysozyme in small intestine)
In Situ Hybridization
• DNA or RNA
distribution
• Target specific
nucleotide probe
– Radioisotope, biotin,
digoxigenin
• Detected by
autoradiography,
color precipitate,
fluorescence
Parathyroid hormone staining (red-ish color) in
Parathyroid gland
Fluorescence Microscopy
• Light source (UV or laser)
• Dyes which when excited emit in the visible
region of the spectrum
• Confocal microscopy
–
–
–
–
Laser light source
Scan across sample and optically section
Useful on live samples (monitor real time events)
Non-destructive (in that it can be used on live
cells)
• Easy sample preparation, fast, inexpensive
Not all fluorescent stains require an
antibody
Phalloidin staining
of actin
Acridine orange
DNA – yellow; RNA - orange
Autoradiography
• Rely on the use of radioactive tag to follow molecule
–
•
•
•
•
•
32P, 35S, 3H
Radioactive probe (amino acids, nucleotides)
Specimen must be alive to monitor metabolic events
Can be used with other stains
Slide dipped in photographic emulsion & developed
Silver grains where radioactive material is present in
sample (AgBr
Ag (precipitate))
• Semi-quantitative
Electron Microscopy
•
•
•
•
Electron beam as source (l ~ 0.005 nm)
Operate under vacuum
Electromagnets instead of glass lenses
Detect by fluorescent screen or photographic
emulsion
• Destructive
• Two major types
– Transmission (TEM) and scanning (SEM)
TEM
Cellular ultrastructure
Bright portion – e- pass through
Dark portion – e- absorbed
or scattered
Sections – 50 – 150 nm
Fix – glutaraldehyde
Stains (e- dense; heavy metals)
Osmium tetroxide (OsO4) – lipids
Uranyl acetate, Lead citrate –
non-specific (surface
adsorbed)
Ruthenium red – complex
carbohydrates
SEM
• Surface ultrastructure
• Fixed, dried, coated with
gold before imaged
• e- reflected from surface
– Results in 3-D like image
SEM image
TEM image
Interpreting EMs
• Use internal rulers as guides
– Plasma membrane ~ 10 nm wide
– Nucleus between 3-10 mm
– Ribosomes between 15-30 nm
• Unique organelle features
– Mitochondria and Golgi
• Look for largest objects first
Problems & Pitfalls
• Shrinkage – fixation and embedding
– Appearance of empty spaces
• Empty spaces due to loss of material
– Improper fixation and dehydration
• Wrinkling
• Precipitation of stains (may appear as dark spots all
over the section)
• Do not memorize images - learn morphological
criteria
• Do not rely on color
Millette’s Laws
• Law #1 – In any given image, if two objects
look the same they are the same.
• Law #2 – In any given image, if two objects
look different they are different.
Goblet cells in Intestine
PAS
H&E
A Tale of Two H&E Stains
2-dimensional
appearance vs.
3-dimensional
structure