CHAPTER 8 - HISTOLOGICAL METHODS
Photoconversion of Fluorescently Labeled Profiles for EM
(Source: S. Wilson)
Labeling cells with DiI
1. Fix embryo for 2-4 hr in 0.1 M Pipes (disodium salt) buffer, 2 mM EGTA, 1 mM MgSO4 and 3.5% paraformaldehyde (or formalin). Adjust pH to 6.95 with concentrated HCl. To this solution add a few drops of glutaraldehyde to give a final concentration of about 0.1% (this last step can be omitted if the preparation is not destined for EM).
2. Wash in 0.1 M PO4 buffer (pH 7.4) and dissect as much as possible to expose the cells that you want to label.
3. Dissolve diI in dimethyl formamide (DMF) or some other organic solvent and allow to dry on glass.
4. Using a sharp tungsten needle, pick up some of the diI crystals (they stick easily to the needle). Put this needle into the bathing solution close to the embryo and using a second needle, pick off a crystal. The diI is neutrally buoyant and will float in the buffer. Using the second needle, move the crystal onto the cell(s) of interest and leave it in place for several minutes.
5. Remove the crystal carefully.
6. Allow the diI to label the cells. The time will depend upon the duration of fixation and the distance that the diI has to diffuse.
DAB photoconversion
1. Make a slide with a well that can hold the embryo and can be coverslipped, for example, a 64 x 48 mm coverslip onto which two pillars of two or three 22x22 mm cover slips have been glued to form a well which sits between the columns.
2. Put the embryo and a drop of DAB (30 mg in 50 ml of PO4) into the well and place a coverslip on top. This cover slip is held by surface tension and the whole slide can be turned upside down without spilling DAB to allow viewing of the embryo from either side. Moving the top coverslip gently allows the tissue to be rotated.
3. Illuminate the labeled cells in the DAB solution with a compound microscope using the normal filters for exciting and viewing diI. The fluorescence fades after a few minutes and a brown reaction product appears. As soon as this happens, remove the specimen by adding a drop of buffer to one side of the well, wash it well, and fix it in 3% glutaraldehyde (buffered to pH 7.2) for 2-3 hr. Do not photoconvert the profiles too long as this leads to spreading of the reaction product beyond the labeled cell and gives poor EM. The reaction product also intensifies when it is osmicated so even if you can barely see the profiles with the light microscope, you should have no problem finding them with EM.
This photoconversion is the make or break step. Two problems tend to arise:
a) Nothing photoconverts. Try the following. Label the cells as brightly as possible, make sure no extra filters are in the light path, use a strong fluorescence light source and use as high a magnification (and numerical aperture) as possible. Allow a longer transport time so that the profiles get as bright as possible. Try using fresh DAB and increasing the concentration to 50 mg/50 ml. Increase the time allowed for photoconversion. You also want your tissue to be as superficial as possible; removing skin and muscle etc. helps.
b) Everything photoconverts and the background is high. Make sure that the DAB is freshly dissolved (or at least not too old). DAB frozen as a concentrated solution eventually seems to lose its potency. Try lowering the concentration of DAB. Try lowering background fluorescence by keeping the application site out of the field of view, and removing the condenser lens (which reflects epi-fluorescence).
7. Photograph and draw the preparation.
8. Osmicate (1%) for one hr, then use a standard EM protocol to get the preparation into plastic. Under a good stereomicroscope you will be able to see the photoconverted cells in the embryo even after it has been embedded in plastic. This helps considerably when you try to find the profiles during sectioning.
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