Rev.9/30/97
Sections are cut at 30-50 um on vibratome into .1 M phosphate buffer (PB).
Sections can be mounted with PB onto a slide, coverslipped and photographed, and then returned to PB.
Prepare about 100 mls of .05 M Tris Buffer pH 8.5, preferably freshly made.
Prepare DAB solution of 1.25 mg/ml by dissolving an aliquot of 12.5 mg/250ul from Finger freezer into 10 mls of .05M Tris pH 8.5.
Dissolve well and wrap container in foil to protect from light.
Select section to photoconvert and place into .05 M Tris to wash 1-10 minutes.
Mount section onto a slide and using a PAP pen draw a circle around it to contain DAB solution.
Apply DAB solution to cover tissue being careful to not create a large puddle which will cause the objectives to get wet.
Slide may be incubated at 4 degrees celcius for up to 10 minutes to facilitiate conversion.
Visualize cell(s) of interest with the flourescent scope and go to the 40x objective.
Photocoversion is performed with the rhodamine filter for about 10 minutes. During the conversion, check to see that the cell remains in focus.
After 10 minutes, most if not all of the flourescence is oxidized and the slide can be removed.
Check section under brightfield scope to determine success.
Sections that have converted cells can be transferred to 2% Glutaraldehyde after washing for 3 x 10’ in PB..