Photoconversion Of Fluorescently Labeled Profiles For EM

(Source: S. Wilson from Zebrafish Book 5th Edition)

Labeling cells with DiI

1. Fix embryo for 2 4 hours in 0.1 M Pipes (disodium salt) buffer, 2 mM EGTA, 1 mM MgSO4 and 3.5% para¬formalde¬hyde (or formalin). Adjust pH to 6.95 with concentrat¬ed HCl. To this solu¬tion, add a few drops of glutaraldehyde to give a final concen¬tration 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 possi¬ble to ex¬pose the cells that you want to label.

3. Dissolve diI in dimethyl forma¬mide (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 solu¬tion close to the em¬bryo and using a second needle, pick off a crystal. The diI is neu¬tral¬ly 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 crys¬tal 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 rotat¬ed.

3. Illuminate the labeled cells in the DAB solution with a compound mi¬cro¬scope using the normal filters for excit¬ing and viewing diI. The fluo¬rescence fades after a few minutes and a brown reaction product ap¬pears. 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% glutar¬al¬dehyde (buffered to pH 7.2) for 2 3 hours. Do not photoconvert the pro¬files too long as this leads to spread¬ing of the reaction product beyond the la¬beled cell and gives poor EM. The reaction product also intensi¬fies when it is osmicated so even if you can barely see the profiles with the light micro¬scope, 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 fluo¬res¬cence light source and use as high a magnifi¬cation (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 concentra¬tion to 50 mg/¬50 ml. Increase the time allowed for photo¬conversion. You also want your tissue to be as superfi¬cial as possible removing skin and muscle etc. helps.

b) Everything photoconverts and the background is high. Make sure that the DAB is freshly dis¬solved (or at least not too old). DAB frozen as a concen¬trated solution eventually seems to lose its potency. Try lower¬ing the concen¬tra¬tion of DAB. Try lower¬ing back¬ground fluorescence by keep¬ing the application site out of the field of view, and remov¬ing the con¬denser lens (which reflects epi-fluo¬rescence).

4. Photograph and draw the prepara¬tion.

5. Osmicate (1%) for 1 hour, then use a stan¬dard EM protocol to get the prepa¬ration into plastic. Under a good stereo microscope, you will be able to see the photocon¬verted cells in the embryo even after it has been embed¬ded in plastic. This helps consider¬ably when you try to find the profiles during sectioning.