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SAMPLE'S PREPARATION

DYES INTO CELLS AND TISSUES

Some fluorescent probes such as DAPI (a DNA dye) generally are not transported through the plasma membrane. Thus, introduction of a dye, or loading, can pose a problem. Often addition of a detergent (saponin, Tween-20, DMSO, etc. ) can be used to facilitate introduction of the dye into living cells. Chromatin dyes can be introduced, and the tissues stabilized, by a pretreatment of 30-50% EtOH or by freezing on dry ice (good for bacteria). This treatment, however, kills the cells. For plant tissue, DAPI pretreatment with EtOH is almost always necessary.of a charged moiety to a fluorophore (fluorescent portion of the molecule) often renders the fluorescent dye permeable. Addition of an acetate to fluorescein (to make fluorescein diacetate, FDA), or a methyl ester group to any of a number of dyes can make a useful dye for living systems. Endogenous esterases cleave acetoxymethyl ester (AM) groups, leaving a non-permeable, but fluorescent dye. These types of dyes are good indicators of cell viability.


Summary:

• Detergent treatment can facilitate introduction of dyes.
• Ethanol (30-50%) can make cells permeable, facilitating dye entry.
• A slight vacuum treatment can be used to remove air pockets, and facilitate dye introduction.
• Gentle abrasion of surfaces.
• Excising and utilizing very small samples; thus increasing surface to volume.

SPECIMEN MOUNTING

One of the major problems in the use and examination of fluorescent microscopic specimens is the tendency of fluorophores to lose fluorescence or quench when they are excited by a light source. Free radicals generated during fluorochrome excitation are responsible for this problem. Several different chemical agents have been used to scavenge free radicals and thus preserve the brightness of the specimens. The pH of the final mounting medium can also make a large difference in the quenching rate.
The steps which I follow in the final stages of fluorescence specimen preparation are as follows:

• After the final rinse of the specimen in PBS, the specimen is dipped in distilled or DI water to get rid of excess salts.
• All excess liquid is removed from the specimen with filter paper or Kimwipe by capillary action. Do not let the specimen dry out!
• A small drop of mounting medium (usually a PBS/Glycerol mix with an antiquenching agent added), several commercial ones are available) is placed on the specimen. A coverslip is carefully lowered onto the drop of mounting medium in such a way to prevent the formation of bubbles.
• Small pieces of filter paper (Whatman #1) are placed around the edge of the coverslip to absorb excess mounting medium. If your specimen is tissue culture cells or frozen tissue sections, this should not harm them. This does two things: a) it allows the coverslip to come as close to the specimen as possible and reduces the depth of the field. You will get less focus aberration if you do this judiciously. b) the coverslip will be a lot less likely to float around on the slide and move which could specimen damage. The surface tension between the coverslip and slide will be much higher. You will also be much better able to seal your coverslip to the slide.
• Seal the edge of the coverslip with clear nail polish and let it dry. If you have pulled off enough excess mounting medium in the previous step, the nail polish will dry much more quickly and not mix with the mounting medium (somewhat of a mess).

If you are using oil immersion, the previous two steps are going to help you in the following ways:

• If you have excess mounting medium around, the oil may mix with it and cause major quenching. By pulling off the excess medium, you lower the likelihood of this problem. Sealing the coverslip will also help a lot.
• Immersion oil has a high surface tension. If your specimen is not properly mounted and sealed, the oil is going to pull on the coverslip during focussing and will cause specimen translation. If it is floating around on a puddle of mounting medium which has a lower surface tension, it will be pulled right off the specimen and destroy it.

It is imperative that you CLEAN UP YOUR SLIDE before you observe it on the fluorescence microscope.


MOUNTING MEDIUM

Mounting media can be prepared in the lab or commercially available ones can be purchased from several of the companies dealing in fluorescent probes.
Mounting medium can be made with 9 parts of glycerol and 1 part PBS. The pH should be adjusted to between 8.5 and 9.0. This pH has been found to be optimal by many investigators in preventing fluorescein and rhodamine quenching. pH's above and below this range will lose fluorescence much more quickly.
A small amount of an antiquench agent or free radical scavenger may also be added to the mounting medium as an added precaution.

Some of these are:

• p-phenylenediamine
• propyl gallate
• ,4-Diazabicyclo (2,2,2)-octane (DABCO)
• Ascorbic acid (That's right folks, vitamin C!)
• Mowiol or Gelvatol