1-HISTOLOGY AND ITS METHODS Flashcards
is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye
Microscope
Single lens
Magnifying Glass
Simple Microscope
Systems (multiple) of lens
Used in Laboratories
Compound Microscope
is the ability of a microscope lens or optical system to produce separate images of closely positioned objects
smallest detail a microscope can resolve
Resolving Power
ability of the microscope to distinguish details. To
detect 2 objects as different objects.
Resolution
ability of microscope to see small objects seem larger
Magnification
(EYE VS INSTRUMENT RESOLUTION)
Human eye
0.2 mm
(EYE VS INSTRUMENT RESOLUTION)
Bright-field microscope
0.2 μm
(EYE VS INSTRUMENT RESOLUTION)
Scanning Electron Microscope (SEM)
2.5 nm
(EYE VS INSTRUMENT RESOLUTION)
Transmission Electron Microscope (TEM) - Theoretical
0.05 nm
(EYE VS INSTRUMENT RESOLUTION)
Transmission Electron Microscope (TEM) - Tissue Section
1.0 nm
(EYE VS INSTRUMENT RESOLUTION)
Atomic Force Microscopy
50.0 pm
Distance Between Resolvable Points
Human Eye > Bright-field microscope> SEM> TEM (Theoretical) > TEM (Tissue Section) > Atomic force Microscopy
: 1 x 10 ^-1000 in M
Micrometer
1 x 10 – 9000 in M
Nanometer
The most used microscope of students and researchers
Bright-field Microscope (LIGHT)
the one providing source of light
light source
allows you to adjust the intensity of the light coming into the object of interest
condenser lens
holds the microscope slide that you are viewing
stage
further magnify the image that you
see in the ocular lens
objective lens
The eyepiece, where we observe our specimen. Has a magnification of 10x.
Ocular lens
Scanning Total Magnification
4x * 10x = 40x
Low Objective Lens Total Magnification
10x * 10x = 100x
High Objective Lens Total Magnification
40x * 10x = 400x
Oil Immersion (OIO) Total Magnification
100x * 10x = 1000x
enables examination of unstained cells and tissues and is especially useful for living cells
Phase Contrast Microscope (light)
Allows quantification of tissue mass
Interference microscope
Useful for assessing surface properties of cells and other biologic objects
3D Image
Differential Interference Microscope
Object will appear bright in a dark background
Is useful in examining autoradiographs, in which the developed silver grains appear white in a dark background.
Dark-Field Microscope
Clinically it is useful in examining urine for crystals and in demonstrating specific bacteria particularly Treponema pallidum.
Beneficial in Microbiology: allow you to appreciate the shape of the bacteria (rods or curve rods shape) and their movements.
Dark-field Microscope
Indicator of syphilis
Treponema pallidum
is a sexually transmitted infection (STI) that can cause serious health problems without treatment.
Syphilis
Indication of cystinuria
Piatos like appearance of crystals
Cysteine Crystals
is an inherited disease that causes stones made of the amino acid cystine to form in the kidneys, bladder, and ureters.
Cystinuria
Used to display autofluorescent molecules (Fluorochromes) such as vitamin A and some neurotransmitters
Fluorescence Microscope
act as stains to view antibody and antigen complex
Fluorochromes
Widespread application is in the detection of antigens or antibodies in immunocytochemical staining procedures
Antibodies can either be polyclonal or monoclonal to detect antigens in the tissue.
Fluorescence Microscope
-Tolerant to antigen epitope change since these heterogenous antibodies are directed against different types of epitopes in an antigen.
-prone to cross-reactivity
Polyclonal
More specific than polyclonal antibodies. Monoclonal antibodies are binding specific or single epitope type in an antigen unlike with polyclonal antibodies.
-with minimal or less chance of cross-reactivity.
Monoclonal
The direct fluorescence antibody test of brain tissues from animals suspected to be infected with rabies.
Positive dFA: Apple green fluorescence
Negative dFA: No bright objects
- the one utilized to detect the
presence of rabies antigen in the tissues. This is particularly called Anti-rabies Antibodies
Reagent antibodies
against the rabies antigen
Anti-rabies antibodies
utilized to label the antibodies to visualize if there is really a binding that happened.
Fluorochromes
if the antigen is present in the tissue. Indicating infection of rabies.
There is a binding
if there is no antigen present in the tissue.
There will be no binding
Orange fluorescence
Acridine Orange
Yellow fluorescence
Auramine O
Bright red fluorescence
Rhodamine B
Apple green fluorescence
Fluorescein Isothiocyanate (FITC)
Using one antibody to detect antigen.
Direct Fluorescence Assay
Using two antibodies to detect antigen
Indirect Fluorescence Assay
Allows visualization of a biologic specimen in three dimensions (3D configuration)
It combines components of a light optical microscope with a scanning system to dissect a specimen optically allowing you to visualize the minute organism
Confocal Scanning Microscope
uses quartz lenses with an ultraviolet light source.
The image depends on the absorption of UV light by molecules in the specimen
Ultraviolet Microscope
may achieve a resolution of 0.1 m and resembles the workings of a spectrophotometer;
results are usually recorded photographically.
is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides
Ultraviolet Microscope
It is also useful for detecting proteins that contain certain amino acids
Also use to determine quantitatively the amount of DNA and RNA in individual cells.
Ultraviolet Microscope
Identify crystals to identify the exact dxs
It uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light
Polarizing Microscope
(POLARIZING MICROSCOPE)
located Between the light source and the specimen.
Polarizer
(POLARIZING MICROSCOPE)
located between the Ocular and the
Objective lens.
Analyzer
has an additional feature as compared to the Light microscope. Which is the addition of 11 filters
Polarizing Microscope
can provide morphologic and analytic data on cells and tissues:
Electron Microscope
Two kinds of EMs
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
uses the interaction of a beam of electrons with a specimen to produce an image. Enables you observe structures found in the inner portion of the organism, it’s organelles.
Transmission Electron Microscope
[T for through (nakikita through the specimen)
Transmission Electron Microscope
the electron beam does not pass through the specimen but is scanned across its surface. Allows you to appreciate its appendages.
Scanning Electron Microscope
[S is for surface (nakikita lang surface ng specimen)
Scanning Electron Microscope
PREPARATION OF TISSUES FOR STUDY
Fixation
Dehydration
Clearing
Infiltration
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labeling
To preserve cell and tissue structure in a life like manner.
Small pieces of tissues are immersed in a solution called fixative after removal from the body
Fixation
best general fixative
10%neutral buffered formalin
Self Digestion
Autolysis
Preserve tissue in a life-like manner
To avoid tissue digestion by enzymes present within the cells or bacteria
Fixation
Fixative ratio
20:1
Remove intracellular and extracellular water. To prepare it in absorbing solutions used in subsequent steps
Tissue is transferred through a series of increasingly concentrated alcohol solutions called as dehydrating agents , ending in 100%, which removes all water
Dehydration
Most common series (DEHYDRATION)
70%, 95%, 100% (absolute alcohol)
Small specimen (DEHYDRATION)
30% - pataas
Why is it in an increasing concentration? (DEHYDRATION)
to avoid tissue distortion
most commonly used alcohol in dehydration
Ethanol (Ethyl Alcohol)
Also known as Dealcoholization
removal of alcohol from the tissue
Clearing
Alcohol is removed in the tissue by immersing in a clearing agent.
Solutions makes tissues clear
Clearing or Dealcholoization
also known as Impregnation
filling the spaces between tissue to make the tissue firm to facilitate cutting
Infiltration
most commonly used for impregnation
Paraffin wax
Tissue is then placed in melted paraffin until it becomes completely infiltrated with the substance.
To facilitate cutting, make tissue stable, and fill spaces
Infiltration or Impregnation
tissues that are fresh and stained. Used
siya for living cells
Vital stain
ang purpose is to remove the unbound
antibodies. Diba, we have a single epitope of rabies Ag, so yung mga other Ab don’t have any Ag to bind to kasi iisa lang naman yung rabies Ag. Dahil naka bind yung anti-rabies Ab sa Ag even after washing, andun pa rin. So ang matitira na lang would be the Ab na nagbind sa rabies Ag. So ang result would be positive.
Washing
medium used for large hollow organs
Celloidin
medium used for brain tissue
Wet Celloidin
medium used for eye organ
Dry Celloidin
used for delicate specimens such as prostrate tissue
Gelatin
Also known as casting
The paraffin- infiltrated tissue is placed in a mold with melted paraffin and allowed to harden
The medium used in infiltration of tissue is the same medium used for this.
Embedding
contains the paraffin infiltrated tissue and
the paraffin mold
Tissue block
Trim the edges to create a perfect block to fit in the microtome
The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome
Trimming
cuts the tissue block into thinner section
Microtome
Most common, invented by Minot
Rotary Microtome
– more dangerous because the knife is the one that is moving across the tissue block
Sliding microtome (Adams)
Rocking microtome
Trefall
obsolete and replaced by cryostat
Freezing microtome (Queckett)
Cut the tissue block into thin films
Tissue block is sliced into thin films using a microtome which are placed on glass slides and allowed to adhere, deparaffinized, and stained.
Section-Cutting
thin films that has impression of the tissues
Tissue ribbon
straighten the tissue ribbon
Water Bath
remove excess paraffin and prepare for staining
Oven
Why is dewaxing important?
Because paraffin wax is not miscible to the stain. If paraffin wax is is not removed, the stain will have difficulty to penetrate the tissue.
The concentration of alcohol in this step is in decreasing concentration. From absolute alcohol going down to up to 70% alcohol
Rehydration
Methods of staining have been devised that not only make the various components conspicuous but also permit distinctions to be made between them.
Staining
cell components with a net negative charge (anionic), they have affinity and stain more readily with basic dyes. (ex: nucleic acid, nucleus)
Basophilic
cationic components, they stain more readily with acidic dyes (ex: proteins)
Acidophilic
Commonly used staining technique
Hematoxylin and Eosin (H&E)
stains DNA, RNA-rich portions of
cytoplasm, and matrix of cartilage. It produces a dark blue or purple color. Color of the nucleus would be blue-black
hematoxylin
acts as a counterstain, meaning it’s a single dye applied separately to distinguish additional features more. It stains the cytoplasm pale pink
eosin
special stains used for lipids
Sudan dyes
used for carbohydrates in the demonstration of glycogen
Periodic acid-Schiff (PAS)
used for nucleic acids
Feulgen technique
DNA and RNA
Methyl Green Pyronin (MGP)
Stained tissue slides are mounted with a cover slip using a mounting media
Mounting
Purpose of mounting
to cover and protect the tissue, to avoid fading of the stain
can be natural or synthetic
Mounting Medium
(MOUNTING MEDIA)
is used in the lab
Synthethic Medium
(MOUNTING MEDIA)
– include Canada balsam from Abies balsamea
Natural Medium
higher refractive index (1.518); avoid fading and protection
Cover slip
resinous mounting media and the most commonly used mounting medium
Canada balsam
Canada balsam refractive index
Refractive index: 1.54-1.55
slides are labelled on the frosted areas with assigned tissue numbers or codes called as “accession number”
Labelling
(FLUID TO TISSUE RATIO)
Dehydration
10:1
(FLUID TO TISSUE RATIO)
Clearing
10:1
(FLUID TO TISSUE RATIO)
Infiltration
25:1
Is a method for localizing newly synthesized
macromolecules in cells or tissue sections
It makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues
Autoradiography
With either______________________, autoradiography permits unique studies of processes such as tissue growth or cellular pathways of macromolecular synthesis
TEM or light microscopy
indicate the cells or regions of cells in which specific macromolecules were synthesized prior
Silver grains
Cells can be grown from newly explanted tissues or as long-established lines and can be examined in the living state by __________
Phase-contrast microscopy
It allows the direct observation of the behavior of living cells
Cell and Tissue Culture
refers to a preparation of cells and tissues that are obtained directly from a tissue or organ and placed in a clear dish under sterile conditions
Primary cell cultures
refers to a process by which certain changes, often related to oncogenes, can promote the cell immortality
Transformation
has been widely used for the study of the metabolism of normal and cancerous cells and for the development of new drugs
Cell culture
is also useful in the study of microorganisms that grow only within cells such as viruses, mycoplasma, and some protozoa
Cell culture
Cervical cancer cells from a px later identified as__________, who died from a dsx in 1951, were used to establish one of the first cell lines, _______, which are still used in research or cellular structure and function throughout the world.
Henrietta Lacks; “HeLa cells”
can be used to confirm the identity of the stained material such as glycogen, DNA, or RNA
Enzyme Digestion
removing of something to confirm the presence of something particularly RNA or DNA.
Enzyme Extraction
Enzyme extraction of dna
potassium dichromate and will inhibit digestion, and should be avoided
Fixation
enzyme extraction of dna
deoxyribonuclease, 0.2 M Tris buffer, pH 7.6, distilled water
Reagents
Enzyme extraction of dna
Results after staining with Fuelgen method:
Test Section
DNA - negative
Enzyme extraction of dna
Results after staining with Fuelgen method:
Control Section
DNA -red
Methyl green will stain your DNA _____
green
Pyronin will stain your RNA ___
red
a tissue section known to have RNA and DNA.
Control Section
If there is NO green and red staining the control section, your control section and stain may be ________
deteriorated
Enzyme extraction of rna
fixation
: potassium dichromate and mercuric chloride will inhibit digestion and should be avoided
Enzyme extraction of rna
reagents
RNAse, distilled water
Enzyme extraction of rna
Results after staining with methyl green-pyronin method:
Test section:
RNA-negative ; DNA-green
Enzyme extraction of rna
Results after staining with methyl green-pyronin method:
Control section:
RNA-red ; DNA-green
Stain the glycogen with Periodic acid-Schiff (PAS)
Color:
Magenta
enzyme that removes RNA in the cell leaving the DNA, when stained by the Methyl Green-pyronin, the result would be RNA negative and DNA Green.
RNAse
digests glycogen
Amylase
high in homogentisic acid
absence of homogentisic acid oxidase causes
Alkaptonuria
Used to look into the activity of enzymes, where exactly in the cell or tissues does this enzyme do its activity or its function (localization of the enzyme)
Used to detect enzymatic products
Uses frozen tissue sectioned in a specialized microtome called cryostat also known as freezing microtome (the conventional counterpart of cryostat, it is like a rotary microtome inside a refrigerator)
Histochemical Techniques
Enzyme classes
phosphatases,
dehydrogenases,
peroxidases
remove phosphate groups from macromolecules
phosphatase
transfer hydrogen ions from one substrate to another
Dehydrogenases
promotes the oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide
Peroxidases
Detects iron storage diseases (hemochromatosis/ hemosiderosis)
Detects which type enzyme has missing/decreased activity
Perl’s Prussian blue reaction
Used to detect glycogenosis and mucopolysaccharidosis
PAS-amylase and alcian blue reactions
Used to detect sphingolipidosis
Lacking enzyme activities results to the accumulation of a certain substance that is needed to be converted by an enzyme. That is why precursor substances are stored and fail to undergo metabolism, hence, accumulation.
Reactions for lipids and sphingolipids
Using primary antibody to detect presence of an antigen
Direct Immunohistochemistry
Using secondary antibody to detect the binding of the primary antibody with the antigen
Indirect Immunohistochemistry
Detecting a mic and an infecting microorganism using complementary nucleotidases
Hybridization Techniques
is based on specific reactions between an antigen or antibodies labeled with visible markers, often fluorescent compounds for light microscopy and gold articles for TEM
Immunohistochemistry
Primary Antibody
we can detect the presence of an antigen through the labelled primary antibody
Direct IHC
primary (not labelled) and secondary Antibody (labelled)
It is the secondary antibody that is labelled that denotes the presence of the antigen through detecting the antibody-antigen complex.
Indirect IHC
Highly sensitive compared to direct technique
Gives more intense result than direct technique
Indirect IHC
2 types of antibodies used in immunocytochemistry
Polyclonal Ab
Monoclonal Ab
Mixture of heterogenous which are usually produced by different B cell clones in the body of an immunized animal
Two of the different B cells are against the antigen but they produce antibodies that attack different epitope
Polyclonal Antibodies
Are generated by identical B cells which are clones from a single parent cell (immortalized Ab-producing cell lines)
Monoclonal Antibodies
an immortalized cell line capable of producing monoclonal antibodies.
hybridoma
APPLICATIONS OFIMMUNOCYTOCHEMISTRY
Prognostic markers in cancer
Tumors of uncertain histogenesis
Metastasis
Response to treatments
Infections
Neurodegenerative diseases
Muscle diseases
Brain trauma
allows the specific identification of sequences of DNA or mRNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe
Commonly performed with nucleic acids in solution
Hybridization
solution of nucleic acid are applied directly to cells and tissue sections
In situ hybridization (ISH)
the label that is attached to identify the nucleic acid sequence
Reporter Molecule
Methods that will be used to view the result depends on the type of reporter molecule:
Fluorochromes
Radioisotopes
Chemiluminescent
Fluorochromes – fluorescent microscope
Radioisotopes – autoradiography
Chemiluminescent – emission of light or Chemiluminescence assay
Several nucleotide probes used in ISH:
Oligonucleotide probes
Single- or double-stranded DNA probes
RNA probes
Labels used for complementary probes:
radioactive isotopes (e.g., 32P, 35S, 3H)
digoxigenin
biotin
a specifically modified nucleotide
digoxigenin
(a commonly used covalent multipurpose
label)
biotin
fluorescent dyes that are combined with nucleotide, making it possible to visualize multiple probes at the same time
Fluorescence in situ hybridization (FISH) procedure
is used to simultaneously examine
chromosomes, gene expression, and distribution of gene products such as pathologic or abnormal proteins
Fluorescence in situ hybridization (FISH) procedure
Many steps in tissue processing, slide preparation, and staining can introduce minor artifacts such as spaces and precipitates that are not normally present in the living tissue and must be recognized
Interpretation of structures in tissue sections
read using Fluorescence Microscope, the result will be in fluorescence.
Fluorescent labels
can be read using Colorimetric Assay- result is indicated by a colored change.
Enzyme label
read using Radioimmuno Assay technique
Rage reactive particles label (Iodine 125)
active sites of antigen. Site where antibodies are binding the antigen
Epitope
is the one that is being detected, the antigen in which the primary antibody is binded
Desmin
: probe nucleotide sequence should be complementary to the sequence of the unknown.
Hybridization Positive
probe is not complementary to the sequence
Hybridization Negative
read the result using Autoradiography
32p – labelled probe
Detected by black areas on film
Radioactive Reporter
: read the result using Colorimetric Detection
Biotin– labelled probe
Detected by changes in color
Biotin-Avidin Reporter
read the result using Chemiluminescent Assay
Acridinium – labelled probe
Detected by capturing emission of light
Chemiluminescent Reporter
orange probe (LSI 21) is locus specific for
chromosome ___
21
____ probe (LSI 13) is locus specific for
chromosome 13
green
