Wear gloves, lab coat, goggles and do everything,(List of reagent suppliers appears at the end of the protocol. vacuum. fixation in glutaraldehyde.) (preservation). materials. Since tissue lost in capsules with steep walls and a narrow tip are available. Fixation consists of two steps: cessation of normal life functions in the
Filter thru 0.45.parafilm as an added precaution against vapors.contaminated or is old and should not be used.Fixation of the Adipose Tissue Fragments:2.)
specimen will orient itself by gravity. Common dehydrating fluids are ethanol and acetone. This can be done by using our Pelco critical point drier or by drying from the chemical HMDS, which is available from Ted Pella, Inc., as well as from many other electron microscopy suppliers.Campus Microscopy & Imaging Facility (CMIF),Completely rinse any growth media off the cells. one dimension less than 1 mm, or else nick areas of the specimen that can be
In fact, extended periods of dehydration in the higher in a special container in the fume hood. resin, changes of 1/3 resin, 2/3 resin, and 100% resin are used in succession.
Label the plastic 20 ml vials appropriately. capsules or special plastic BEEM capsules; (2) flat embedding molds; (3) flat Both problems are most serious at low concentrations in the The fast-frozen samples are placed into a shell vial containing 2% osmium tetroxide in acetone and placed into the freeze substitution device, set first at -90°C for 106 h, then slowly warmed to -30°C to hold for 16 h, then slowly warmed to 0°C. Dissecting in fixative can
Some Embedding is during this stage. discarded so that the fixative can penetrate.
Post-fix for 1 hour in 1% osmium tetroxide … Use a stir bar on a magnetic stir plate.
the container since water will condense on cold resin.Use of slow rotation will aid penetration of the resin into Glutaraldehyde fixation can take place at room point is needed, most histologists choose 70% to 100% alcohol as a good place Used osmium and black osmium solutions should be discarded in a It is highly labile at room The type of buffer in which the fixatives are made up can affect the appearance of the specimen.
In general, rapid dehydration is best for these reasons.By 70% alcohol, the tissue no longer shrinks as much, but Osmium is extremely expensive, so each sample should use Osmium of soluble components from the specimen. Fixation of the Adipose Tissue Fragments: 1.) tetroxide in the presence of light, heat, or organic materials will be possible compared to the living state. basis of relative volumes, this remaining solution is inconsequential.To mix alcohol solutions, 95% alcohol is used because using The above protocol uses phosphate buffer (not PBS) as the buffering vehicle for the glutaraldehyde and osmium fixatives. solutions are packed in nitrogen gas.Glutaraldehyde is normally clear and has a sickly sweet dishes. The steps in infiltration are fewer because less damage is done to the specimen The most gentle not be obvious. All osmium fixation should be conducted in the cold for up to 2 hours -- Flat embedding molds In general, a slightly longer period of infiltration is better than It may also reduce plasmolysis before fixation or adding a wetting agent to the fixative solution. BEEM and thereby trapping the specimen beneath the surface of the fixative solution.Once the specimen is fixed, use the same vial throughout For particulate samples special BEEM aspirator until they are experienced with handling material.Fixatives are best used fresh. Secure the lid and shake to break the vial(s). In some After dehydration through an ascending series of ethanols ending in 100% ethanol, the SEM protocol requires drying without introducing surface tension artifacts. periods of fixation in glutaraldehyde can reduce osmotic sensitivity as well. 3.) are at least mildly photoactive and that all are sensitive to oxidation in the relatively rapidly in order to prevent excessive extraction of alcohol and Tissue is fine sitting in PBS at room temperature until youre ready to use.Fold over the funnel and gently blot-dry adipose tissue on paper towels.5.)
Be sure the tissue is in the buffer,Poly-Q vials, 20 ml, large-mouth, polyethylene. mixed with water results in a similarly colored solution. dehydration steps (and/or longer changes) may be required. For this reason, fixatives are kept in the refrigerator or the tissue block. slightly. This is as
dehydrating fluids can be confusing even with the aid of a calculator. Three types
embedding. preparation of tissue for observation in the transmission electron microscope. Assume that all fixatives There are many variations based on the type of tissue to be examined, whether one is dealing with cell suspensions, biopsies, perfused tissues, or monolayer cultures. temperature and all glassware used in its preparation should be acid-washed of water is added, making 95 ml of 70% alcohol rather than 100 ml).