CHAPTER 5: CMB Techniques (pero Embryo talaga
to)
A.Microscopy
Dissecting microscope
-binocular microscope with x10-x50 magnification range
with good working distance between objective lens and
specimen
-provides a 3D image (not inverted) accurate perception
of depth (good for microinjection and microsurgery)
For opaque specimen:
Incident Lighting (light is shone down
onto the specimen from the light source) is
used.
For living specimen:
So as to not overheat them a fiber optic
light guide (provides a cold but bright
illumination) is used.
For transparent specimen:
A transmitted light base is used.
Compound Microscope
-magnification range of x40-x1000; upper limit of
magnification is set by the wave nature of light which
prevents resolution of points closer together than about
0.2 um
-Image is inverted; used for sections or wholemounts
(that are small enough to be transparent)
Optical Techniques
**ordinary transmitted light is used to examine stained
sections (through histological stains or histochemical
rxn)
**wholemounts should be thin to be transparent
Phase Contrast Microscopy
-converts small differences of refractive index within
the specimen into large differences of light density
-for in vitro live cells, isolated gametes, & small
transparent embryos
Differential interference contrast (aka Nomarski
Optics)
-converts small differences of refractive index
into an apparent difference in height when perceived by
the observer.
- provides a sharp resolution of a particular
optical section of specimen so as one focuses up and
down through the specimen.
-provides clear visualization of single cells
within small transparent specimen.
Fluorescence
-absorbing light of specific energy and emits lower
energy (longer wavelength)
-depend on visualizing the location of a fluorochrome
within a specimen
-fluorescent antibody staining, fluorescent in situ
hydbridization, and visualization of fluorochromes as
labels to subsets of cells in a specimen.
-Excitation spectrum- how the intensity of emitted
fluorescence varies with excitation wave length
-Emmission spectrum- how the intensity of emitted
fluorescence is distributed across wavelength spectrum
Fluorescence microscope parts:
Lamp- (before mercury arc, now LED) shines excitation
beam on to specimen
Filter- selects a narrow excitation wavelengty band
suitable for the fluorochrome in use. Alternative sets
are used for (one each) different fluorochrome.
Dichroic mirror- reflects excitation beam to specimen A;
reflects excitation beam with low cut off and emits
above cutoff
Dark Field Microscopy
-only specimen that scatter light extensively are visible
and appear as bright points due to illumination from a
very oblique angle
-signal is caused by accumulation of silver grains due to
photographic emulsion coating the section
Polarization Microscopy
-alters plane polarization of light (bifringence); appears
as bright patches
Confocal microscopy
-for thick wholemounts
-uses laser for illumination so that excitation is achieved
with just a single wavelength characteristic of that laser
-illuminates one point at a time thus high quality
- all points are scanned -> fluorescence of each point is
recorded -> image of section is reconstructed
-light above or below the plane is dispersed
contributing less to the signal
-image is stored in a computer
Multiphoton microsocope
-better than confocal
-light source is of less energy than the required energy
to excite the fluorochrome thus only two or more
photons needed to strike a fluorochrome.(this can only
happen at the point of focus where the photons are
very dense)
Electron microscopy
-provides more magnification than required for
identifying cell types and embryos thus it’s rarely used
for devt biology.
Although SEM provide vivid 3D views of wholemounts
and is usuallty used for visualizing cell arrangements.
Image Capture
-images are recorded by photography (1990) and by
charged couple devices. (present)
its detection chip consists of array of pixels each
of which can be filled up with electrons
charge of each pixel is proportional to the
intensity of light falling on it which is read out in
sequence by a detector and is converted to an image,
-8 bit image- represented by no.’s 0-255
-24 bit image- represented by one 8 bit
number for each of three colors: red,
green, and blue
Most sensitive functions only in B&W, and can
collect long exposures from very faint specimens; able
to count single photons only
Less sensitive ones can collect images fast
enough to display on a video screen and records them
real time.
An advantage of digital image collection is signal/noise
ration can be improved by taking several exposures and
then averaging them thus signal will be same in each
exposure and therefore, noise is uniform and low.
Time Lapse
-useful for morphogenic movements of cells, organ
cultures and embryos
-individual images are taken at intervals and are played
at shorter intervals
-stage control is required so that the specimen will stay
at the field of view
-environmental chamber which keeps the embryos at
37C and 5% CO2 (for avian and mammalian embryos,
and organ culture)
Histological methods
Fixation- specimen is ‘killed’ and made mechanically
robust to withstand osmotic shocks and certain amount
of handling.
Fixatives:
Formalin -> most common; gas formaldehyde that react
with amino or sulfuhydril groups to denature or form
cross links.
Glutaraldehyde -> fixative that has two aldehydes and is
very effective for intermolecular cross linking.
Organic solvents and acids- denaturating and
precipitating.
Dehydration- removing H2O by passing the specimen
through a series of ethanol baths in increasing conc.
- Essential because a direct transfer from water
to pure alcohol causes tissue damage that arises
from mixing forces by solvents.
Once H2O is removed it is immersed in xylene, a solvent
miscible with wax. It is then embedded in a solid
supporting material usually paraffin wax (60C) to allow
it to be cut into thin sections.
(NOTE: heat and organic solvents may damage proteins
and nucleic acids thus compromising immunostaining or
ISH)
(NOTE (nenemen): Paraffin wax should not be used if
sections to be cut is less than 5um. Certain plastics may
be used if specimen is to be cut at 1um but most
plastics are not compatible with immunostaining and
ISH)
Sectioning- use of
Microtome- where block passes repeatedly
across a very sharp knife each time advancing
the block by a few microns, and produces
ribbons.
Parts of ribbons are mounted on slides to be
stained, or to be processed for immunostaining
or in situ hybridization.
Cryostat- microtome operated in a cooled
chamber. The block mounted here may not be
necessarily fixed but if frozen rapidly in a
medium with high conc. sucrose.
-not good for serial section because it does not
form ribbons.
B. Studying gene expression by molecular biology
methods
Biochemical methods- accurate quantitive measure but
not anatomical information.
In situ methods- accurate anatomical information but
limited quantitive measure.
**It is desirable to do both as the transcriptiom of a
gene does not guarantee later translation to protein
and the presence of protein in a particular place does
not guarantee that it has been synthesized there.
Laser capture- molecular analysis on specific cells taken
from microscopic sections.
Stage series- wherein biochemical methods are used by
examining groups of embryos of different
developmental stages
METHODS FOR MESSENGER RNA:
1. Reverse transcription polymerase chain
reaction (RT-PCR)
- Standard for measuring and detecting mRNA
RNA is reverse transcribed into
Total RNA extracted cDNA (complementary DNA) using
from sample reverse transcriptase
The DNA sequence between
primers is amplified by repeated
Two oligonucleotides are added.
cycles of synthesis, melting, and
hybridization.
Reaction mxtures is run on a gel.
DNA bands are visualized by
ethidium bromide staining.
If reaction worked: BAND (corresponding to the size of the region of cDNA
between primers) APPEARS
If reaction did not work: NO BAND APPEARS;
thus no specvific c target DNA in the sample
**what is examined is the accumulated
concentration at the end of the reaction when
plateau is reached the method is at best only as
semiquantitive.
Loading control- guarantees the presence of similar
amount of RNA in each sample.
Includes: Positive and negative sample, and a “no
reverse transcription” control in case contaminating
DNA generates a signal.
** cycles should not be more than 35 as it will only
detect biologically insignificant trace levels of mRNA.
 2. Real time PCR
-rate of formation of the amplified product is
monitored in real time
Example is: dye SYBR Green- fluoresced brightly
when bound to double stranded but not single
stranded DNA. Increase fluorescence means
high buildup of amplified product.
3. Ribonuclease analysis
-most sensitive for detection of specific mRNA
but is rarely used today.
4. Northern blotting
-oldest and most sensitive technique
4. Microarrays
-enable examination of large numbers of gene
products simultaneously;
-contain a subset of the complete genome
-used in the early stage of a project to identify
the genes likely to be involved in a particular
system or process.
-has two basic types: (used for nucleic acid
hybridization)
a. cDNA array
-consists of a regular
arrangement of closely spaced spots,
each of a different cDNA, arranged in a
rectangular array on glass slide
-long synthetic oligonucleotides
are used

-can show the number and size of mRNA’s b. Affymetrix oligonucleotide array
where there are several splice variants formed
- large number of short
from the same gene.
oligonucleotides are synthesized
directly on sides.
-several oligonucleotides
Extracting total mRNA
from specimen represent sequences from one gene

a probe is prepared by
extracting RNA from the
embryo or tissue sample
separating via gel
electrophoresis on
denaturating agarose gel.

Reverse transcription of mRNA

to cDNA
Contents of gel are blotted on
a hybridization membrane.

labeling of cDNA with

fluorescent dye and hybridizing
Membrane is hybridized by it to the microarray under
specific probe which is suitable conditions
labeled with a chemical tag
that is detected by enhanced
chemiluminescence reaction.
Array is scanned by a chip
reader (fluorometer in this
case) that measures the
Chemical tag is visualized by fluorescence from the dye
autoradiography or bund to each spot.
phosphorimager.
 Amount of florescence ~ amount of cDNA thus
~ amount of mRNA
In developmental biology, microarrays are used
to make comparisons. (two embryonic stages or
between cells with and without inducing factor)
The two different probes are labeled with
different fluorochromes (Cy3- green, Cy5- red) and
are mixed before hybridization. Genes whose
expression did not change will produce yello while
green for upregulated and red for down regulated.
5. Deep Sequencing Method
-RNA seq
-ability to measurethe composition of
entire scriptome in a cDNA sample.
-each known exon that is encountered is
identified by comparison with the known
genome of the organism then the frequency of
each product is counted relative to others
METHODS FOR PROTEIN
Proteomics- set of techniques to identify individulal
unknown proteins from a complete mixture
-employed at beginning of experiment to identify
protein change in expression level during a particular
developmental stage
Mass spectrometry- used to identify proteins by ionizing
and volatizing substanceto acquire accurate protein
molecular weight (which can be calculated from the k
nown amino acid composition of each polypeptide
represented in a genome sequence)
Tandem mass spectrometry- protein is digested with
trypsin into a number of peptides (for 1st cycle), then
each peptide is broken up by ion bombardment (for 2nd
cycle).
Immunochemistry- measures protein sample based on
the antibody concentration that is directed against the
protein (may be monoclonal or polyclonal)
***all methods below are examples of
immunochemistry method***
1. Western Blot
- Shows content of specific protein.
Protein of sample are extracted
and separated on an acrylamide
gel
Protein content is blotted onto a
membrane
Membrane is incubated using a
primary antibody specific for the
target protein
Rseult is incubated with secondary
protein conjugated with the enzyme
horseradish peroxidase specific for
the primary antibody.
Rseult is incubated with substrate
micture and the blot is exposed to
Xray film or placed in a
phosphorimanger.
-phosphorescence is recorded in the position
where the antibody is bound and correspond to
the position and quantity of target protein
sample.
2. Immunoprecipitation (not true precipitation)
-isolates the protein recognized by specific
antibody
-used to find whether a particular protein is
being newly synthesized in an embryo or tissue
sample
- nonradioactive mode is usually followed by
western blot
Sample is incubated in radioactive amino
acids then with a specific antibody to form
amm immune complex
Immune complexes are isolated by
incubating it with protein A bound to
agarose beads.
beads are washed to remove conatminants
and are boiled to seaparate antobody from
protein
SDS protein gel is used to separate proteins
by molecular weight.
Gel is dried, and visualizzed by
autoradiography or phosphorimanger.
 3. Chromatin immunoprecipitation
-involves
a. shearing chromatin fragments of about 500
nucleotides in length then
b. immunoprecipitating with an antibody to a
protein of interest (chromosomal protein)
c.sample is deproteinized and the DNA is
analyzed by RT-PCR
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C. Study of gene expression by in situ methods
-reveal spatial domains of gene expression in a
specimen
Specimen that are small and transparent enough- in situ
on wholemount, otherwise, in situ on sections
1. In situ hybridization
-reveals the ragions of specimen where specific
mRNA is present.
-chemistry is same with northern blot
- antisense probe is synthesized in vitro and
includes an extra chemical group (attached on
one of the nucleoside triphosphates used for
synthesis) that is recognizable
commercialized specific antibody.
-chemical group examples: digoxigenin (a plant
sterol) and fluorescein.
-for wholemount in situ hybridization specimen
should be permeabilized by protease or
detergent to enable probe molecules to enter
the cells.
n
Specimen is washed
l desired cell clone is isolated it can be grown and
anzyme linked anti-probe antibody
with enzyme is added
complex is placed in a substrate
mixture yielding a colored insoluble
precipitate at the site of rxn
Fluorescent lSH- if substrate used is fluorescent;
more than one probe is visualized at a time by
using different chemical tags on the probes
thus, detected by different antibodies.
**Radio labeled probes- highly sensitive
**Color rxn- can be watched and be stopped as
it reaches desired density.
2. Immuno staining
-adjuvant- where protein is mixed when it is
bound to be injected into an animal
-antigen- target of specific antibody; usually a
protein
-epitope- part of antigen recognized by
antibody
a. Polyclonal antibodies- usually made in
rabbits
-consist of products of several clones of B
lymphocytes each making an antibody from
different antibody gene
-contain unrelated antibodies recognizing
unrelated antigens, and other non antibody
by a proteins thus, the need of purification via
column of protein A (isolates igG
antibodies) or affinity chromatography.
b. Monoclonal. Antibodies
-made by immunizing mice fusing their spleen
with a human tumor cell called myeloma.
(hybrid is called hybridoma; capable of antibody
production and growth wthoit limit in vitro)
-clones of fused cells are screened and once the
medium harvested as a source of a particular
monoclonal antibody.
-don’t have high affinity for their antigen as
polyclonal antibodies
Immunostaining in wholemounts provide a 3D view
of the location of the antigen in specimen while in
section procieds a higher resolution.
Common principle:
- Incubate with specific antibody, wash, incubate
with 2ndary anti body (commercially available).
- If fluorescent (simpler) 2ndary antibody, use
fluorescent microscope directly.
- If enzyme linked (more sensitive), it must be
incubated with substrate to produce
colorimetric rxn.
When precipitate is formed, non aqueous mounting
medium (used in Enzyme-linked) is preferred to
dehydrate specimen because they have higher
refractive index thus rendering specimen more
transparent.
Fluorescent method is impossible to be dehydrated thus
it is not permanent because immune complexes will
easily dissociate. However, it is the best method to use
in looking at more than one antigen because each
fluorochrome can be examined separately in its own
fluorescence microscope.
3. Reporter genes
- Encode easily detectable product to:
o Monitor events in developing organism
o Indicate activity of transgene
o Label a particular cell type
o Indicate activity of specific intacellular
signaling pathway
- Analyze regulatory domains of genes
o Short sequence from putative
regulatory region is attached to a
reporter gene and introduced to
embryo as transgene. If sequence is
active expression of reporter and
domain can be visualized.
- Examples:
a. lacZ coding for B-galactosidase
-B galactosidase-enzyme capable of hydrolyzing
B galactosides
-usually used in situ
-detected at extremely low levels (highly
sensitive)
-X-gal is used (5-bromo-4-chloro-3-indoyl-B-
Dgalactoside) as substrate.
*when hydrolyzed from ga;lactose X
produces green blue insoluble
precipitate
-perdurance- possible to find regions in embryo
where B galactosidase is present but mRNA is
not because of itsstability.
-B-geo= fusion of B galactosidase with product
of neomycin resistance gene.
b. Green Fluorescent protein-
- from jellyfish (Aequora Victoria); remains
active when fused with other proteins
-easy to visualize in living specimen thus
allowing real-time examination of labeled cells
-fluorescence is lost after fixation
c. Luciferase
-catalyzes breakdown of luciferin in the
presence of ATP, a reaction that emits light.
-emmission spectrum peaks in the green but
extends to longer wavelengths, which penetrates
tissued easily
-phosphorescence is measured by luminometer
-other luciferase from, sea pansy (Renilla) uses
another substrate (coelenterazine) has blue
emission and is used as normalization control
for firefly luciferase.
D. Microinjection
-used to introduce genes, transgenes, and inhibitors and
lineage label (substance visible that identifies a specific
cell lineage)
-equipment depends on the size of target cell; usually
mounted on microscope to allow visual control of the
injection.
-micromanipulator- holds the needle and reduce
manual movements
-needle is made up of glass tubing. -hydrophobic, thus dissolve in lipid
membranes and well retained in
-substance may be introduced to needle my capillary progeny
action or via syringe at near end
-seen using fluorescent microscope
-needle is connected to controller by a flexible tube
-removed by organic solvents
-Controller types:

- Pressure device- applies sharp pulses of

pressure and forces a small volume out of the sharp 2. Intacellular labels
end.
a. Fluorescent dextrans
-Iontophoretic device- applies an electric field
-polymer of glucose, solublke in water,
across the needle and causes migration of appropriately
and metabolically inert; 10, 000 Mol.
charged molecules out of the tip
Weight.
Xenopus- under dissecting microscope
-FDA (fluorescein dextran amine) and
Other smaller specimens- compound microscope but its RDA (rhodamine dextran amine)
optics will be arranged so that image is not inverted
-remain confined to the cell bc it can’t
E. Cell Labeling methods diffuse through lipid membrane

-used for fatemapping- shows normal destiny of embryo -visible by conjugating to a

regions in the course of development fluorochrome

-label cells into which other substances like mRNA and -gixable by conjugating to lysine (readily
antibodies have been introduced reacts to fixatives like aldehyde)

-without growth (Xenopus and zebrafish), substances b. Horseradis peroxidase

don’t become diluted and can remain visible for days
-catalyze oxidation of various substrate
1. Extracellular labels by hydrogen peroxide

Vital dyes: Nile blue and neutral red -diaminobenzidine – normally used
substrate; converted to a brown
-not lineage labels insoluble material stable to paraffin wax
histology.
-reasonable intensity of color; does not
produce toxic effects -can be visualized in formalin fixed
wholemounts via frozen sections.
-labels whole specimen by application
in medium or to label specifc regions of 3. Genetic labels
embryo by local application of small
block of agar impregnated with the dye. -incorporated into genome and becaome
replicated evry cell cycle and are not diluted
-cheap, quick, and simple
a. Mouse glucose phosphate isomerase isozyme system
Carbocyanine dyes: DiI (red) and DiO (green)
-depend on electrophoretic activity
-applied using extracellular variant of
the microinjection
-visualized only by biochemical analysis; low spatial
resolution

b. Retroviruses

-RNA that carry reverse transcriptase which makes a

DNA copy of the viral genome that can then integrate
into the chromosome of the cell, thus remaining in the
cell progeny.

-Replication-incompetent virus- lacks genes that are

necessary for assembly of virus particles; unable to
produce virus or unable to lyse the cell. Thus, is
propagated by packaging cell lines which contain the
missing viral genes and are able to complement the
functions missing from the virus.

-retrovirus used for cell labeling usually contain lacZ

gene

-Long terminal repeat- strong promoter sequence