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Wax Embedding Of Specimens And Preparing The Slides |
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| This process uses flammable materials and solvents as well as some of the materials being corrosive or irritant to eyes, please familiarise yourself with hazard warnings on labels or in safety data, use solvents in well ventilated areas and avoid contact with the materials and breathing of fumes. |
I must first say I am a novice at wax embedding having had a very steep learning curve in order to do one specific job in sectioning honey bee wings along the plane of the wing in order to understand how the wing veins may modify light transmission when the wings are optically scanned using a slide scanner. This page has been assembled after having read many web sources and some very helpful Emails from my friends who are better versed in microscopy than myself. It has been generalized in order to deal with wider aspects than just my original reason for getting involved.
All referances to water imply distilled or de-ionised water, unless stated otherwise.
If we are starting with a whole bee as our initial specimen, we will have to reduce it to manageable pieces that will either fit on to a slide or be able to be cut into thin sections using a microtome and the sections mounted and stained if necessary.
In order to arrest biological decay and preserve the structure of the material, we need to render the cells inert and possibly strengthen soft tissue in order to retain shape. Formaldehyde is commonly used for this and is often dissolved in water, when it is known as Formalin, it causes covalent cross-linkages to be added between proteins, thus strengthening the material as well as rendering the tissue sterile.
Other reagents can be used...
- Acetic Acid at 5% to 10% strength (Strong White Vinegar)
- Various Alcohols at 91% to 100% strength.
- Copper Sulphate, (I have seen 2%, 5% and 10% recommended).
- Lactic Acid at 10% strength
- Copper Acetate 2% to 5%
- Potassioum Dichromate at 1%,2% and 5%
Timing of fixation will depend on the thickness of the specimen and the strength of fixative, and may range from as little as 15 minutes for hevy metal fixatives not listed here, alcohol fixation takes up to 12 hours and formalin may require much as two whole days. After the desired time has elapsed, the excess fixative should be removed by soaking the specimen in two changes of water.
In some cases specimens will have been pickled in aclohol for transport purposes, so this fixation step will not be required.
Water has to be removed from the tissue of the specimen by repeated soaking in ethyl or Isopropyl Alcohol solutions of progressively stronger strength. for our embedding purpose we will use Isopropyl Alcohol (also called Isopropinol).
To dehydrate our specimen soak it in 70% Isopropyl Alcohol for two hours. Then transfer to 91% Isopropyl Alcohol for another two hours, blot the specimen on a tissue and progress to 100% Isopropyl Alcohol (absolute Isopropyl Alcohol) for two hours, followed by two hours in a change of 100% Isopropyl Alcohol, with a final change of again 100% Isopropyl Alcohol for 12 hours or overnight. For consistency, blotting can be performed at every change as it instills good habits.
The now dehydrated specimen is saturated with alcohol that is not miscible with paraffin wax, so we will have to remove it with something that is solute for paraffin wax and compatible with Isopropyl alcohol. The sustance we are going to use for this purpose is Toluene, the step is known as clearing due to some tissues becoming more transparent during the process.
having removed the Alcohol and replaced it with toluen, we must now remove Embedding: This step allows a supportive substance to infiltrate a specimen followed by hardening. Paraffin (wax) is commonly used to fill in spaces in tissue while it is in the liquid state (warm), then the entire block solidifies at room temperature. This permits the original shape of the specimen to be maintained through subsequent processing. Plastic is often substituted for paraffin. Sectioning: The supported specimen is cut into thin slices. This is usually done on a sharp metal blade mounted in a microtome to ensure that sections are of a known and constant thickness, usually around 25 microns. Floating out Affixing: The section is flattened onto a glass slide to permit it to adhere to the glass. This often requires a type of ?glue? to keep the section on the glass through further processing. Glass is used in making permanent slides for a variety of reasons. These include the fact that glass resists scratching, it can be cleaned better than plastic, and the optical properties of glass allow for better resolution than plastic. drying removal of wax and Re-Hydration Staining: The slide with the affixed section is placed in an appropriate stain that will result in visual contrast between structures in the finished slide (see staining page). This step is often preceded by from the slide and specimen. Mounting and Coverslipping: A mounting medium with optical properties similar to glass is used to permanently embed the specimen in resin or syrup and to permanently attach the glass coverslip and seal it's edges.
Printed from Dave Cushman's website Live CD version
Written... Revised... Upgraded...
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