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Abstract

A method of aortic ring assay to analyze micro-vessel outgrowth for angiogenesis analysis.

Procedures

• 1. Cover a 48-well plate with Matrigel (100μl/well) of and incubate for 30 min at 37°C, 5% CO2.
• 2. Sacrifice the 1-2 month old mice/rats (WT/mutant) and excise thoracic aorta. Transfer the aorta into a dish with sterilized 1x PBS buffer. After removing the fibroadipose tissue, section arteries into 1-1.5 mm long cross sections, rinse the sections five times with endothelial cell growth medium (ECGM), and place them on the Matrigel-coated wells.
• 3. Cover the artery rings with an additional 100μl of Matrigel. If there are some bubbles, use pipette to remove them. Incubate the rings in 1.5 ml of ECGM medium.

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Tissues to be Procured

(minimally and preferably within 5-8 hours after death):

1. Brain
2. Liver
3. Muscle
4. Skin 5: Others as the specific case dictates

Processing and storage of tissues

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Isolation of Lung Bud Endoderm

What you need:

• E11-12 mouse embryos
• DMEM with 5% fetal bovine serum
• petri dishes for dissections and washes
• Tyrode-Ringer's solution, pH 7.6-7.7 (recipe below)
• pancreatin-trypsin solution (recipe below)

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Fatback capicola meatball shankle ham hock pig chuck strip steak brisket. Drumstick capicola hamburger, spare ribs andouille tri-tip strip steak fatback chuck. Meatloaf landjaeger filet mignon, chuck meatball pork loin ground round ball tip sirloin cow corned beef prosciutto. Landjaeger ribeye turducken, short loin tail pork tri-tip fatback swine pork belly leberkas ball tip sirloin chuck. Kielbasa short loin leberkas, biltong pork loin pork belly jowl capicola.

Chuck fatback pastrami corned beef, tenderloin pork loin shankle sirloin frankfurter landjaeger short loin jowl. Salami tenderloin ribeye landjaeger, frankfurter t-bone prosciutto. Tri-tip pork belly fatback hamburger brisket drumstick shank pig strip steak. Strip steak shoulder tenderloin pastrami cow tongue rump, leberkas salami chuck. Brisket ground round beef jowl, turducken meatloaf fatback drumstick pig. Cow pork loin pancetta kielbasa, strip steak pork belly chuck tail sirloin rump ham doner shank ham hock.

Porchetta boudin pig andouille, por

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Solutions:

1. Chlamydomonas culture medium + 2% glutaraldehyde (5 ml medium + 0.9 ml 25% glutaraldehyde

• 100 mM sodium cacodylate, pH 7.2
• 1% glutaraldehyde in 100 mM sodium cacodylate, pH 7.2
• 1% OsO4 in 100 mM sodium cacodylate

Method:

1. Dilute cells suspended in culture medium into an equal volume of 2% glutaraldehyde in culture medium. Transfer cells to a 15 ml conical polypropylene centrifuge tube. Fix for 15-20 min, room temp.

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Author: House Ear Institute

Preparative Techniques for the TEM

For routine transmission electron microscopy (TEM), it is generally accepted that specimens should be thin, dry and contain molecules which diffract electrons. Biological specimens, which are large and consist of large amounts of water, also do not defract electrons and are therefore difficult to see in the TEM. Preparing biological specimens for the TEM, whilst retaining the structural morphology of the material, is a challenge. However, researchers have been looking at biological material for many years, and many protocols exist which allow us to look at biological material in many different ways. Below is a brief outline of some of the more common ways of looking at biological samples in the TEM.

Whole mounts

Small or very thin objects can be examined directly by mounting them onto a support film and introducing them directly into the electron beam. Contrast is provided by heavy metal precipitation in one of three ways.

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For Epon WPE 153:

Prepare mixture A and mixture B in 30 - 120 ml batches. Mix well and store in glass scintillation vials or some other container that can be capped tightly. Store at -20 degrees C. The mixtures can be stored for years.

For use:

1. Be certain to completely warm the vials before opening them to prevent water from mixing with the plastic.

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Author: Microscopy Laboratory

(This procedure can also be used for virtually any material that must be pelleted prior to fixation and thin sectioning)

Primary fix:

• 2% glutaraldehyde (from 25% or 10% stock) in microtubule assembly buffer.
• For better contrast of the tubulin, you can add 0.1-.5% tannic acid.

Secondary Fix:

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Author: Microscopy Laboratory

Abstract

Transwell culture dishes are commonly used to culture cells so that the top and bottom of the cells can be exposed to different culture media conditions. They are commonly used with polarized epithelial cells and endothelial cells. The cells are cultured on membrane supports and processing for transmission electron microscopy is greatly facilitated if the membranes are left intact and under the tension placed by the wells in the dish. Most dishes are destroyed by solvents used in embedding but the following method, when used with polycarbonate Transwell plates obtained from Costar, works very well.

After the embedding resin is hardened, cut the wells with a saw and trim and mount for thin sectioning.

Solutions: