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1Slide
Other MethodsOther Methods•Northern blotting – analyzes mRNA expression in
tissue
• In situ hybridization – Visualizing gene activity (mRNA) directly in fixed cells or tissues
•Western blotting – analyzes proteins expressed in tissue
• Immunohistochemistry (IHC) or Immunocytochemistry (ICC) – labels proteins in fixed cells or tissues
•Northern blotting – analyzes mRNA expression in tissue
• In situ hybridization – Visualizing gene activity (mRNA) directly in fixed cells or tissues
•Western blotting – analyzes proteins expressed in tissue
• Immunohistochemistry (IHC) or Immunocytochemistry (ICC) – labels proteins in fixed cells or tissues
2Slide
Northern BlottingNorthern Blotting
Purpose: Can tell us if a particular gene is being expressed in a specific cell or tissue type or if
there are changes in levels of expression-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
•Isolate mRNA from cells
•Separate mRNA with gel electrophoresis
•Blot (transfer) the mRNA to a membrane
•Label mRNA you are interested in (probe with a complementary sequence that binds your mRNA of interest)
Purpose: Can tell us if a particular gene is being expressed in a specific cell or tissue type or if
there are changes in levels of expression-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
•Isolate mRNA from cells
•Separate mRNA with gel electrophoresis
•Blot (transfer) the mRNA to a membrane
•Label mRNA you are interested in (probe with a complementary sequence that binds your mRNA of interest)
3Slide
In situ HybridizationIn situ Hybridization
Purpose: Allows us to visualize gene activity directly in tissues or cells
-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
• Tissues may be cut into thin sections or entire embryo may be adhered to a microscope slide
• mRNA probes are labeled with a dye or enzyme that makes a colored precipitate
• Tissues are imaged with a microscope
Purpose: Allows us to visualize gene activity directly in tissues or cells
-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
• Tissues may be cut into thin sections or entire embryo may be adhered to a microscope slide
• mRNA probes are labeled with a dye or enzyme that makes a colored precipitate
• Tissues are imaged with a microscope
4Slide
Western BlottingWestern Blotting
Purpose: Detects levels of proteins being produced in a particular cell or tissue
-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
• Isolate proteins
• Separate proteins with gel electrophoresis
• Blot (transfer) the proteins to a membrane
• Label protein you are interested in (antibody that binds specifically to your protein)
• Bands can tell if protein is present and if there are different molecular forms of it
Purpose: Detects levels of proteins being produced in a particular cell or tissue
-----------------------------------------------------------------------------------------------------------------------------------
General Procedure:
• Isolate proteins
• Separate proteins with gel electrophoresis
• Blot (transfer) the proteins to a membrane
• Label protein you are interested in (antibody that binds specifically to your protein)
• Bands can tell if protein is present and if there are different molecular forms of it
5Slide
Example of Western Blotting
EXNE
Tur
key
NB
Tur
key
BR
Female Male
EXNE EXNEEXNE
Zebra finch anterior pituitaries
Breeding Breeding Not Breeding
Not Breeding
Prolactin Isoforms in the Anterior Pituitary of Zebra Finches
The lane on the extreme left is the ladder (mol wt marker). Tissue extracts in the remaining lanes are labeled with a prolactin antibody. Some birds demonstrate a second, slightly heavier band of prolactin. Because it is heavier, it migrates more slowly, and shows prolactin exists in two forms in these birds; the heavier prolactin has been modified by adding a sugar group.
6Slide
MicroscopyMicroscopyThe idea behind any type of microscopy is to
see inside the tissue and/or cell.
What we see and how well we see it, depend upon:
1. Type of microscope
2. Tissue preparation
3. Tissue staining
The idea behind any type of microscopy is to see inside the tissue and/or cell.
What we see and how well we see it, depend upon:
1. Type of microscope
2. Tissue preparation
3. Tissue staining
7Slide
Types of MicroscopesTypes of Microscopes
Light Microscope: Resolution of about 0.2m
Good for routine staining
Fluorescent microscope:
Fluorescent dyes are excited by specific wavelength of light and re-emit it at higher wavelengths (lower energy).
(ex: FITC – a very common fluorescent dye – is excited at 488, emits at 525nm - looks green)
Filters eliminate stray light, so the image is sharper
Light Microscope: Resolution of about 0.2m
Good for routine staining
Fluorescent microscope:
Fluorescent dyes are excited by specific wavelength of light and re-emit it at higher wavelengths (lower energy).
(ex: FITC – a very common fluorescent dye – is excited at 488, emits at 525nm - looks green)
Filters eliminate stray light, so the image is sharper
8Slide
Confocal MicroscopeConfocal Microscope
Uses a laser to excite fluorescent dye in tissue or cell one spot at a time
Precisely positioned pinholes allow only in-focus light to pass through
Images in XYZ planes
Computer takes all images and creates a 3D reconstruction
9Slide
Confocal microscope: 0.2-0.5m resolution3D images can be obtained
http://www.jneurosci.org/content/vol26/issue46/images/data/11870/DC1/Fig._12C-PBC-3D_animation-Ruangkittisakul_et_al.mov
Ion movements can be recorded
]http://www.jneurosci.org/content/vol26/issue46/images/data/11870/DC1/Fig._12A-PBC_calcium_oscillations-Ruangkittisakul_et_al.mov
Confocal microscope: 0.2-0.5m resolution3D images can be obtained
http://www.jneurosci.org/content/vol26/issue46/images/data/11870/DC1/Fig._12C-PBC-3D_animation-Ruangkittisakul_et_al.mov
Ion movements can be recorded
]http://www.jneurosci.org/content/vol26/issue46/images/data/11870/DC1/Fig._12A-PBC_calcium_oscillations-Ruangkittisakul_et_al.mov
Anterior pituitary of a zebra finch. Cells secreting prolactin appear red, while those secreting growth
hormone appear green.
An individual cell in the lower right corner has both hormones and appears yellow. The dark
“hole” in the middle of the cell is its nucleus.
From: Christensen, 2007
Confocal MicroscopeConfocal Microscope
10Slide
Tissue PreparationTissue Preparation
Fresh tissue is thick, mushy, and opaque. Also has active enzymes and bacteria that promote degradation.
3 basic steps:
1. Fixation – stabilizes structure
2. Embedding & sectioning – makes it hard so we can cut thin slices
3. Staining – provides contrast so we can see it easier
Fresh tissue is thick, mushy, and opaque. Also has active enzymes and bacteria that promote degradation.
3 basic steps:
1. Fixation – stabilizes structure
2. Embedding & sectioning – makes it hard so we can cut thin slices
3. Staining – provides contrast so we can see it easier
11Slide
Histological TechniquesHistological Techniques
Fixation: stabilizes structure
stops enzymes
prevents bacterial degradation
(putrefaction)
The most common fixatives cross-link tissue
proteins. An example is formaldehyde.
Fixation: stabilizes structure
stops enzymes
prevents bacterial degradation
(putrefaction)
The most common fixatives cross-link tissue
proteins. An example is formaldehyde.
12Slide
Embedding & SectioningEmbedding & Sectioning
Embedding: Infiltrate tissue with harder substance that is more easily cut into thin sections
1. Resin – plastic, used for very thin sections, 0.5-1µm
2. Paraffin – used for sections >5µm
3. Freezing – used for sections 20-100µm
Embedding: Infiltrate tissue with harder substance that is more easily cut into thin sections
1. Resin – plastic, used for very thin sections, 0.5-1µm
2. Paraffin – used for sections >5µm
3. Freezing – used for sections 20-100µm
13Slide
SectioningSectioningParaffin (frozen similar)
1. Sectioned with a rotary microtome
2. Metal or glass knife or razor blade
3. Sections to 1-2 µm thick
Paraffin (frozen similar)
1. Sectioned with a rotary microtome
2. Metal or glass knife or razor blade
3. Sections to 1-2 µm thick
14Slide
StainingStaining
Staining - introduces contrast to tissue which is otherwise essentially transparent
Common stains: Hematoxylin (looks blue-purple) & eosin (looks pink-red)
Usually hematoxylin and
eosin are used together
(H&E)
Staining - introduces contrast to tissue which is otherwise essentially transparent
Common stains: Hematoxylin (looks blue-purple) & eosin (looks pink-red)
Usually hematoxylin and
eosin are used together
(H&E)
15Slide
HistochemistryHistochemistryHistochemistryHistochemistryCan stain DNA & visualize replicating cellsCan stain DNA & visualize replicating cells
16Slide
DNADNADNADNA
BRd-U - bromodeoxyuridine - analog of thymidine, incorporated when DNA replicates
a. Antibody stains labeled cells
b. Can follow time sequence of cell division
BRd-U - bromodeoxyuridine - analog of thymidine, incorporated when DNA replicates
a. Antibody stains labeled cells
b. Can follow time sequence of cell division
17Slide
Immunohistochemistry (IHC)Immunohistochemistry (IHC)
Advantages
1. High specificity for molecular species
2. Can be used for light, confocal, or EM
Advantages
1. High specificity for molecular species
2. Can be used for light, confocal, or EM
Disadvantages
1. Time consuming & expensive
2. Fixation can interfere with Ab binding
3. Reproducibility - false positives - cross reactivity
4. Difficult to get Abs to small molecules
Disadvantages
1. Time consuming & expensive
2. Fixation can interfere with Ab binding
3. Reproducibility - false positives - cross reactivity
4. Difficult to get Abs to small molecules
Uses antibodies to detect specific molecules in tissue
18Slide
Immuno methodsImmuno methods
1. Direct method - label (dye) is on the antibody1. Direct method - label (dye) is on the antibody
19Slide
2. Indirect method - label is on a secondary antibody, amplifies label. Primary antibody binds to molecule of interest
Second antibody (labeled with dye) against species in which primary was raised - eg, goat anti-
rabbit antibody
2. Indirect method - label is on a secondary antibody, amplifies label. Primary antibody binds to molecule of interest
Second antibody (labeled with dye) against species in which primary was raised - eg, goat anti-
rabbit antibody
Immuno methodsImmuno methods
20Slide
LabelsLabelsFluorescent dyes - FITC, rhodamine, Cy3, Cy5 - different excitation and emission spectra allows double labelingEnzymes (horse-radish peroxidase (HRP) or alkaline phosphatase) give colored precipitate
Fluorescent dyes - FITC, rhodamine, Cy3, Cy5 - different excitation and emission spectra allows double labelingEnzymes (horse-radish peroxidase (HRP) or alkaline phosphatase) give colored precipitate