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Comparing approaches

Techniques


Comparing Approaches

3 ways to detect beta-galactosidase

photo Three different ways to detect the same transgene (LacZ). Livers from mice that received beta-galactosidase-expressing adenovirus were processed for X-gal staining to detect enzyme activity (top), for immunohistochemistry with antibodies against beta-galactosidase (bottom), and for in situ hybridization with a riboprobe specific for beta-galactosidase (LacZ) transcripts (middle). Images are from livers of different animals.

Reporter-gene detection

beta-galactosidase

photo X-gal staining of muscle (top) and lung (bottom) sections from mice that had received beta-galactosidase-expressing AAV vectors. The beta-galactosidase used for gene transfer into lung contained a nuclear-localization signal and therefore is detectable only within nuclei.

GFP

photo GFP expression from viral vectors in eye (top) and liver (bottom). In the eye section, which shows part of the retina, nuclei had been stained with DAPI.

Alkaline phosphatase

photo Detection of alkaline phosphatase activity in lung of a mouse that had received an AAV vector expressing placental alkaline phosphatase. The lung of an untreated control animal is shown in the bottom image.

Firefly luciferase

photo The expression of luciferase can be detected in tissues of living animals after injection of luciferin, resulting in the emission of light. Shown are mice that have received AAV vectors expressing firefly luciferase in muscle (top) and liver (bottom).

Immunostaining

Immunohistochemistry

photo Mouse lymph node stained for B-cells with anti-CD45R antibodies. Nuclei are counterstained with hematoxylin in blue.
photo Immunohistochemical staining of T-cells with anti-CD3 antibodies in a mouse lymph node.
GFP Expression of GFP in monkey liver after gene transfer demonstrated by immunohistochemistry.
photo Lung sections of SARS-corona-virus-infected mouse immunostained with antibodies against the viral nucleocapsid (N) protein. Infected cells are visible mainly in the airway epithelia.
photo Anti-BrdU (5-bromo-2'-deoxyuridine) staining of nuclei in intestine (top) and liver (bottom). Animals received BrdU which incorporates into replicating DNA serving as a marker for proliferation.Higher magnification of the liver sample shows two labeled nuclei of a hepatocyte.

Immunofluorescence

photo Expression of transgenic Factor IX from AAV vectors in liver (top) and muscle (bottom) detected by immunofluorescence.
photo Immunofluorescent staining of Kupffer cells in a monkey liver with antibodies against CD68 (red). Nuclei are stained with DAPI in blue.
Ad-DAPI Cells infected with adenovirus were stained with antibodies against Ad showing infected cells in green and nuclei stained with DAPI in blue.
photo Confocal image showing a macrophage in the lung of a mouse infected with SARS-corona virus. Double immunofluorescence labels the macrophage in red with antibodies against Mac-2 and viral protein in green with antibodies against spike. *
MC

Left: Muscle injected with AAV was sectioned and stained with antibodies against CD8 (blue), CD4 (green), and Foxp3 (red nuclei) showing different types of T cells between muscle fibers. Regulatory T cells are positive for CD4 and Foxp3. Right: same section showing in addition nuclei (DAPI stain) in light blue.


Histochemistry

photo
Liver section with infiltrate stained for chloroacetate esterase activity (Leder stain, in red). This enzyme is a marker for granulocytes such as neutrophils.
photo
Histochemical detection of OTC (ornithine transcarbamlyase) activity in liver. Mice deficient in functional OTC received AAV vectors expressing this enzyme. OTC activity was visualized by a histochemical procedure leading to the deposition of lead sulfide within transduced hepatocytes.
GUSB H

Dog spleen stained for endogenous β-glucuronidase (GUSB) activity (red). Absence of GUSB activity causes the lysosomal storage disease mucopolysaccharidosis VII.


Special Stains

Gram stain

photo Gram staining of lung infected with a non-pathogenic strain of Bacillus anthracis.

PAS

photo Periodic Acid Schiff (PAS) staining of kidney section. PAS stains carbohydrates present in basement membranes and brush borders in pink-magenta.

Oil Red O

photo Oil Red O stained liver section. This dye stains fat droplets in bright red.

Trichrome

photo Trichrome staining on aortic root lesion (plaque) showing collagen in blue (cryosection).

Movat

photo Movat pentachrome staining of arteries with plaque formation. Elastic fibers are stained in black, collagen in yellow, ground substance in blue.

Alcian Blue

Alcian Blue - monkey colon Monkey colon stained with Alcian Blue for acidic mucosubstances showing goblet cells.

 


TUNEL assay

photo TUNEL assay on paraffin sections of lung (top, fluorescein label) and testis (bottom, stained with DAB). The TUNEL assay is aimed at detecting fragmented DNA as indicator of apoptotic events. Terminal transferase is used to elongate DNA breaks with labeled nucleotides. Shown are positive controls (left, treated with DNase) and negative controls (right, treated with DNase but without transferase) of tissue sections.

In Situ Hybridization

photo RNA in situ hybridization on paraffin liver sections with a digoxigenin-labeled riboprobe specific for beta-galactosidase transcripts. Shown are livers from a mouse that had received a beta-galactosidase-expressing adenovirus vector (top) and from an untreated control animal (bottom).
photo RNA in situ hybridization on cryosections of mouse liver with a digoxigenin-labeled riboprobe specific for GFP mRNA. Animals had received AAV vectors expressing GFP (top), liver from an untreated animal serving as control (bottom).
photo Fluorescent in situ hybridization (FISH) was used to detect replicating DNA (yellow) of an adenoviral vector in 293 cells. This vector carries the gene for factor VIII and was detected with a labeled FVIII cDNA probe. Nuclei had been counterstained with propidium iodide (red).
photo FISH on cryosections of human heart with a probe specific for Alu repetitive elements. Shown are the Alu stain (top, nuclei in green) and the corresponding DAPI image (bottom, nuclei in blue).

Plastic sectioning

photo Toluidine-blue stained plastic sections (1 Ám) of nasal septum (top) and liver (bottom).

Electron Microscopy

EM negative staining

photo Negative staining of adenovirus. Particles have a diameter of about 100 nm.
photo Negative staining of an AAV vector preparation. Particles have a diameter of about 25 nm.
photo Ebola virus-like particles (VLPs) visualized by negative staining.

EM of cells

photo TEM micrograph of a dendritic cell.
photo Cell with budding vesicular stomatitis virus (VSV).
photo Newly formed adenoviral particles in the nucleus of an infected 293 cell.

Immuno-EM

photo Detection of insulin in pancreatic islet cells by immunohistochemistry using a light microscope (top) and by immuno-electron microscopy with gold-labeled antibodies (bottom, part of nucleus can be seen in lower part of image).

* The confocal image above for Immunofluorescence was published in the Journal of Virology (Hogan et al. 2004, J. Virol. 78: 11416) and is reproduced here with permission.

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