BIOL 465 Histology
Review Sheet
Chapter 1 Methods
• The maximum resolution of the bright-field microscope is 0.2μm. It requires a
very thin slice of tissue.
Explain where “artifacts” come from and why they are a problem.
Upon preparing a histologic slide, an artifact or error would occur. That would be
due to the usage of contaminated reagents and chemicals, handling of improper
equipment like a microtome with a defective blade, or not following the methods of
preparation carefully, as failing to follow proper timing in each stage or carelessly
placing the coverslip. Artifacts are a problem because it will be confusing when
examining the slide, but that is why students have to get known with the most
common artifacts on slides.
Describe how tissues are “fixed.”
Tissues are immersed in a fixative after removing them from the body. The most
commonly used fixative is Formalin and it’s an aqueous formaldehyde solution that
inhibits cell metabolism, prevents tissue degradation, kills pathogens and allows the
proteins to bind, therefore hardening the tissue.
Describe the processes of embedding and sectioning.
After fixation, the tissue is washed and dehydrated in a series of alcohol solutions of
ascending concentration. Then, in a step called clearing, organic solvents like xylol,
that is miscible with alcohol and paraffin would be used to remove the alcohol.
Later, the tissue is embedded in paraffin wax. (Sectioningg)Paraffin is trimmed into
blocks, where a block would be placed in a microtome and cut with a steel knife. The
sections are placed on glass microscope slides.
Describe the general process of chemical staining.
First, paraffin is removed (using xylol). Next, the tissue is rehydrated. Then, the
tissue on the slides is stained, and after that is dehydrated. Finally, it is mounted on
a clear glass slide and covered with a glass coverslip.
Explain how a basic dye works and give three examples of basic dyes.
The molecules of the basic dye are positively charged, therefore it binds to
negatively charged components of cells and tissues (e.g. DNA).
Three examples of basic dyes: Methyl green, methylene blue, pyronin G,
toluidine blue, hematoxylin
Explain how an acidic dye works and give three examples of acidic dyes.
Acidic dyes are negatively charged and bind to positively charged components of
cells and tissues (e.g. proteins).
Three examples: Acid fuchsin, aniline blue, orange G
Those three are used in combinations (e.g. Mallory trichrome technique), to
selectively color different constituents of the tissue(muscle and collagen in this
case).
Which combination of dyes is the most commonly used? Hematoxylin and eosin
(H&E)
,Explain metachromasia and name one type of tissue that exhibits metachromasia.
Metachromasia happens with basic dyes, in which the basic dye molecules are close
together to form dimers or polymers and change the color of the dye from blue to
red or purple. This happens when the concentration of phosphate or sulfate groups
is high, such that cartilage, mast cell granules, and rough endoplasmic reticulum of
plasma cells exhibit metachromasia.
Explain why the periodic acid-Schiff (PAS) reaction is useful in histology.
PAS reaction is useful in histology because it is able to stain molecules rich in
carbohydrates like glycogen, basement membrane(protein), and mucus, as they are
shown in a magenta color.
Explain the difference between immunohistochemistry (IHC) and
Immunofluorescence (IF).
So, in the case of IHC, primary antibody specifically binds to the protein that is to be
studied. To identify the site of binding, enzyme-conjugated secondary antibody is
used, as it’s directed against the primary antibody. A substrate is added to the
secondary antibody and it changes color then the proteins of interest show up in a
new color. To identify that color in the tissue, a light microscope is used.
However, in IF, (just like in IHC), primary antibody specifically binds to the protein
that is to be studied. Then, a fluorophore- conjugated secondary antibody is used. To
identify the protein of interest, a fluorescent microscope is used.
Explain the difference between a primary and a secondary antibody.
A primary antibody binds to the antigen in the sample. A secondary antibody
specifically binds to the primary antibody.
Explain why it is important to know about antigens and epitopes when using
antibodies for IHC or IF.
An epitope is a piece of the antigen(protein) that the antibody recognizes, and it
could be a short sequence of amino acids within the protein, yet it could be hard to
identify because it sometimes includes sequences from different parts of 3D
structure. Also, during fixation, cross-linking of protein molecules occurs, which
prevents the antibody from recognizing the protein of interest. That is why a
process called antigen retrieval is done in the beginning, to get the antigen in its
natural form, through breaking the formaldehyde cross-links.
Explain the difference between a monoclonal and a polyclonal solution of
antibodies.
Polyclonal antibodies bind to multiple epitopes. They are produced by animals,
through taking the protein of interest, purifying it, and injecting it into the animal.
Multiple B cells would be activated, forming clones, and producing the antibodies.
Then, antibody purification from the serum takes place. However, monoclonal
antibodies production is different. First, B cell (that secretes specific antibodies) is
isolated from the animal and is fused with another immortal cell (cancer cell),
forming a hybridoma. So, the specific antibodies produced in the culture medium
are collected. And since the origin is a single B cell, the antibodies are monoclonal
(one type of antibody), thus recognizing one epitope of an antigen.
Explain when you would use hybridization (rather than IHC or IF).
Hybridization is used to detect a specific region of DNA or RNA.
,Chapter 2 Cytoplasm
Describe each of the organelles of the cell.
• Nucleus: largest organelle in the cell
• Plasma membrane: made up of two layers of phospholipids that form the
boundaries of the cell and the boundaries of many organelles within the cell.
• Rough endoplasmic reticulum: it is the region of er that is associated with
ribosomes. And it is the site of protein sorting and synthesis.
• Smooth endoplasmic reticulum: It is the region of er that is not associated with
ribosomes, but it’s involved in synthesis of lipids and steroids. Also, it contains
detoxifying enzymes and stores Ca^2+ and it’s involved with storing and
metabolizing glycogen and formation & recycling of the membrane.
• Lysosomes: are tiny organelles that contain digestive enzymes (e.g. nucleases,
lipases and phospholipases, proteases, phosphatases...). They break down excess
or worn-out parts of the cell by endocytosis.
• Golgi: The cis (forming) face of the Golgi is where the vesicles from the er empty
their content, while the trans(maturing) face of the Golgi is where the vesicles
leave it to different parts of the cell. So, the Golgi is well developed in secretory
cells and it is the site of sorting, secretion, and modification of proteins and lipids.
• Mitochondria: provide most of the energy to the cell by producing ATP.
Mitochondria can move, grow, and divide independently; they have their own
DNA, ribosomes, and tRNAs. Also, they play an important role in apoptosis.
• Peroxisomes: Small organelles that are involved in breaking down of hydrogen
peroxide and other harmful molecules.
Describe the structure and function of the plasma membrane.
The plasma membrane contains glycoproteins(proteins modified with carbohydrate
groups) and glycolipids (lipids modified with carbohydrate groups) that are
expressed on the outer surface of the plasma membrane.
The glycocalyx (cell coat) is the outer surface of the plasma membrane, and it
contains receptor proteins for growth factors and hormones and is used for cell
recognition.
Lipid rafts are subdomains of the plasma membrane that are enriched in
phospholipids, where the membrane at that region is thicker and less fluid. Those
rafts regulate neurotransmission and receptor trafficking.
Explain the importance of intermediate filaments in histology.
Intermediate filaments are highly tissue specific, which allows us to identify cell or
tissue types within a slide. Keratins is one major class of intermediate filaments, in
which keratins are found in hair & nails and epithelial cells. There are other cells
that have intermediate filaments; in the connective tissue there’s vimentin, in
neurofilaments there’s neurons, and in every kind of cell in the body there are
lamins, that are found in the inner surface of nuclear membrane.
, Explain what an inclusion is and how it is different from an artifact.
Inclusions would show up upon examining histological specimens and those
inclusions are not organelles, cytoskeletal components, or artifacts. They are natural
components of the cell, in which they’re cytoplasmic or nuclear structures formed
from metabolic products of the cell. They could be lipofuscin, fat droplets,
glycogen, hemosiderin, or crystalline.
An artifact is an error that occurs due to the usage of contaminated reagents and
chemicals, handling of improper equipment like a microtome with a defective blade,
or not following the methods of preparation carefully, as failing to follow proper
timing in each stage or carelessly placing the coverslip.
Chapter 3 Nucleus
Describe the dyes used to stain chromatin.
Hematoxylin and basic dyes
Differentiate between heterochromatin and euchromatin.
Heterochromatin is a condensed form of chromatin found in the nucleus and it
stains very darkly with H&E. Also, it is condensed DNA that cannot be transcribed.
Euchromatin is another form of chromatin and it stains lightly. It is dispersed and
active for transcription.
Describe static, stable, and renewing cell populations.
Static cell populations consist of cells that no longer divide.
Stable cell populations consist of cells that most of the time do not divide, but may
divide in case of injury.
Renewing cell populations consist of cells that may be slowly or rapidly renewing
that exhibit regular mitotic activity. The slowly renewing populations include
smooth muscle cells, fibroblasts of uterine wall, epithelial cells of the lens of the eye.
The rapid renewing populations include blood cells, epithelial cells and fibroblasts
of the skin, and epithelial cells and fibroblasts of the GI tract
Draw a diagram of the cell cycle, and explain the role of regulatory proteins in the
cell cycle.
Draw a cell in each of the phases of mitosis, and describe what occurs at each phase.
Explain how meiosis is different than mitosis.
• Mitosis is a process where a single cell divides into two identical daughter
cells.
• Meiosis is a process where a single cell divides twice and produce four
daughter cells.
• Mitosis produce two diploid cells that are genetically identical to each other
andand to the original parent cell.
• Meiosis produce four haploid cells that are genetically unique from the each
other and the original parent cell.
• In mitosis there is only one cell division while in meiosis there are two cell
divisions.
• Before meiosis begins the DNA of each chromosome is replicated (during S
phase of cell cycle).
Review Sheet
Chapter 1 Methods
• The maximum resolution of the bright-field microscope is 0.2μm. It requires a
very thin slice of tissue.
Explain where “artifacts” come from and why they are a problem.
Upon preparing a histologic slide, an artifact or error would occur. That would be
due to the usage of contaminated reagents and chemicals, handling of improper
equipment like a microtome with a defective blade, or not following the methods of
preparation carefully, as failing to follow proper timing in each stage or carelessly
placing the coverslip. Artifacts are a problem because it will be confusing when
examining the slide, but that is why students have to get known with the most
common artifacts on slides.
Describe how tissues are “fixed.”
Tissues are immersed in a fixative after removing them from the body. The most
commonly used fixative is Formalin and it’s an aqueous formaldehyde solution that
inhibits cell metabolism, prevents tissue degradation, kills pathogens and allows the
proteins to bind, therefore hardening the tissue.
Describe the processes of embedding and sectioning.
After fixation, the tissue is washed and dehydrated in a series of alcohol solutions of
ascending concentration. Then, in a step called clearing, organic solvents like xylol,
that is miscible with alcohol and paraffin would be used to remove the alcohol.
Later, the tissue is embedded in paraffin wax. (Sectioningg)Paraffin is trimmed into
blocks, where a block would be placed in a microtome and cut with a steel knife. The
sections are placed on glass microscope slides.
Describe the general process of chemical staining.
First, paraffin is removed (using xylol). Next, the tissue is rehydrated. Then, the
tissue on the slides is stained, and after that is dehydrated. Finally, it is mounted on
a clear glass slide and covered with a glass coverslip.
Explain how a basic dye works and give three examples of basic dyes.
The molecules of the basic dye are positively charged, therefore it binds to
negatively charged components of cells and tissues (e.g. DNA).
Three examples of basic dyes: Methyl green, methylene blue, pyronin G,
toluidine blue, hematoxylin
Explain how an acidic dye works and give three examples of acidic dyes.
Acidic dyes are negatively charged and bind to positively charged components of
cells and tissues (e.g. proteins).
Three examples: Acid fuchsin, aniline blue, orange G
Those three are used in combinations (e.g. Mallory trichrome technique), to
selectively color different constituents of the tissue(muscle and collagen in this
case).
Which combination of dyes is the most commonly used? Hematoxylin and eosin
(H&E)
,Explain metachromasia and name one type of tissue that exhibits metachromasia.
Metachromasia happens with basic dyes, in which the basic dye molecules are close
together to form dimers or polymers and change the color of the dye from blue to
red or purple. This happens when the concentration of phosphate or sulfate groups
is high, such that cartilage, mast cell granules, and rough endoplasmic reticulum of
plasma cells exhibit metachromasia.
Explain why the periodic acid-Schiff (PAS) reaction is useful in histology.
PAS reaction is useful in histology because it is able to stain molecules rich in
carbohydrates like glycogen, basement membrane(protein), and mucus, as they are
shown in a magenta color.
Explain the difference between immunohistochemistry (IHC) and
Immunofluorescence (IF).
So, in the case of IHC, primary antibody specifically binds to the protein that is to be
studied. To identify the site of binding, enzyme-conjugated secondary antibody is
used, as it’s directed against the primary antibody. A substrate is added to the
secondary antibody and it changes color then the proteins of interest show up in a
new color. To identify that color in the tissue, a light microscope is used.
However, in IF, (just like in IHC), primary antibody specifically binds to the protein
that is to be studied. Then, a fluorophore- conjugated secondary antibody is used. To
identify the protein of interest, a fluorescent microscope is used.
Explain the difference between a primary and a secondary antibody.
A primary antibody binds to the antigen in the sample. A secondary antibody
specifically binds to the primary antibody.
Explain why it is important to know about antigens and epitopes when using
antibodies for IHC or IF.
An epitope is a piece of the antigen(protein) that the antibody recognizes, and it
could be a short sequence of amino acids within the protein, yet it could be hard to
identify because it sometimes includes sequences from different parts of 3D
structure. Also, during fixation, cross-linking of protein molecules occurs, which
prevents the antibody from recognizing the protein of interest. That is why a
process called antigen retrieval is done in the beginning, to get the antigen in its
natural form, through breaking the formaldehyde cross-links.
Explain the difference between a monoclonal and a polyclonal solution of
antibodies.
Polyclonal antibodies bind to multiple epitopes. They are produced by animals,
through taking the protein of interest, purifying it, and injecting it into the animal.
Multiple B cells would be activated, forming clones, and producing the antibodies.
Then, antibody purification from the serum takes place. However, monoclonal
antibodies production is different. First, B cell (that secretes specific antibodies) is
isolated from the animal and is fused with another immortal cell (cancer cell),
forming a hybridoma. So, the specific antibodies produced in the culture medium
are collected. And since the origin is a single B cell, the antibodies are monoclonal
(one type of antibody), thus recognizing one epitope of an antigen.
Explain when you would use hybridization (rather than IHC or IF).
Hybridization is used to detect a specific region of DNA or RNA.
,Chapter 2 Cytoplasm
Describe each of the organelles of the cell.
• Nucleus: largest organelle in the cell
• Plasma membrane: made up of two layers of phospholipids that form the
boundaries of the cell and the boundaries of many organelles within the cell.
• Rough endoplasmic reticulum: it is the region of er that is associated with
ribosomes. And it is the site of protein sorting and synthesis.
• Smooth endoplasmic reticulum: It is the region of er that is not associated with
ribosomes, but it’s involved in synthesis of lipids and steroids. Also, it contains
detoxifying enzymes and stores Ca^2+ and it’s involved with storing and
metabolizing glycogen and formation & recycling of the membrane.
• Lysosomes: are tiny organelles that contain digestive enzymes (e.g. nucleases,
lipases and phospholipases, proteases, phosphatases...). They break down excess
or worn-out parts of the cell by endocytosis.
• Golgi: The cis (forming) face of the Golgi is where the vesicles from the er empty
their content, while the trans(maturing) face of the Golgi is where the vesicles
leave it to different parts of the cell. So, the Golgi is well developed in secretory
cells and it is the site of sorting, secretion, and modification of proteins and lipids.
• Mitochondria: provide most of the energy to the cell by producing ATP.
Mitochondria can move, grow, and divide independently; they have their own
DNA, ribosomes, and tRNAs. Also, they play an important role in apoptosis.
• Peroxisomes: Small organelles that are involved in breaking down of hydrogen
peroxide and other harmful molecules.
Describe the structure and function of the plasma membrane.
The plasma membrane contains glycoproteins(proteins modified with carbohydrate
groups) and glycolipids (lipids modified with carbohydrate groups) that are
expressed on the outer surface of the plasma membrane.
The glycocalyx (cell coat) is the outer surface of the plasma membrane, and it
contains receptor proteins for growth factors and hormones and is used for cell
recognition.
Lipid rafts are subdomains of the plasma membrane that are enriched in
phospholipids, where the membrane at that region is thicker and less fluid. Those
rafts regulate neurotransmission and receptor trafficking.
Explain the importance of intermediate filaments in histology.
Intermediate filaments are highly tissue specific, which allows us to identify cell or
tissue types within a slide. Keratins is one major class of intermediate filaments, in
which keratins are found in hair & nails and epithelial cells. There are other cells
that have intermediate filaments; in the connective tissue there’s vimentin, in
neurofilaments there’s neurons, and in every kind of cell in the body there are
lamins, that are found in the inner surface of nuclear membrane.
, Explain what an inclusion is and how it is different from an artifact.
Inclusions would show up upon examining histological specimens and those
inclusions are not organelles, cytoskeletal components, or artifacts. They are natural
components of the cell, in which they’re cytoplasmic or nuclear structures formed
from metabolic products of the cell. They could be lipofuscin, fat droplets,
glycogen, hemosiderin, or crystalline.
An artifact is an error that occurs due to the usage of contaminated reagents and
chemicals, handling of improper equipment like a microtome with a defective blade,
or not following the methods of preparation carefully, as failing to follow proper
timing in each stage or carelessly placing the coverslip.
Chapter 3 Nucleus
Describe the dyes used to stain chromatin.
Hematoxylin and basic dyes
Differentiate between heterochromatin and euchromatin.
Heterochromatin is a condensed form of chromatin found in the nucleus and it
stains very darkly with H&E. Also, it is condensed DNA that cannot be transcribed.
Euchromatin is another form of chromatin and it stains lightly. It is dispersed and
active for transcription.
Describe static, stable, and renewing cell populations.
Static cell populations consist of cells that no longer divide.
Stable cell populations consist of cells that most of the time do not divide, but may
divide in case of injury.
Renewing cell populations consist of cells that may be slowly or rapidly renewing
that exhibit regular mitotic activity. The slowly renewing populations include
smooth muscle cells, fibroblasts of uterine wall, epithelial cells of the lens of the eye.
The rapid renewing populations include blood cells, epithelial cells and fibroblasts
of the skin, and epithelial cells and fibroblasts of the GI tract
Draw a diagram of the cell cycle, and explain the role of regulatory proteins in the
cell cycle.
Draw a cell in each of the phases of mitosis, and describe what occurs at each phase.
Explain how meiosis is different than mitosis.
• Mitosis is a process where a single cell divides into two identical daughter
cells.
• Meiosis is a process where a single cell divides twice and produce four
daughter cells.
• Mitosis produce two diploid cells that are genetically identical to each other
andand to the original parent cell.
• Meiosis produce four haploid cells that are genetically unique from the each
other and the original parent cell.
• In mitosis there is only one cell division while in meiosis there are two cell
divisions.
• Before meiosis begins the DNA of each chromosome is replicated (during S
phase of cell cycle).
