Photoconversion
Animation of a 200-um thick photoconverted hippocampal slice of an aged (11-mo old) mouse. This was done by a photooxidation procedure (see below) in which flourescently labeled granule cells were oxidized under a Zeiss 10x objective. Clearly, the cells show certain degree of degeneration / loss of dendritic trees (provided by http://www.neurodigitech.com). Copyright (C) 2009 Neurodigitech All Rights Reserved. Note: this vide was generated by Neurodigitech, LLC. Photoconversion protocol for DiI labeled sections: 1) Prepare DAB 50mg/ml in phosphate buffer, pH7.2 in advance. 2) Pre-soaking of the sections in the diaminobenzidine (DAB) solution at RT for 5 minutes. 3) Mount the section on a glass slide with a Petri dish, add the DAB to the dish, and cover the tissue with glass coverslip. 4) Photo-oxidize as needed. Higher numerical aperture and magnification results in faster conversion but the trade-off is the limited field of view (FOV) of the section and this generally will produce a relatively higher level of background oxidation. You can first start with a plan-neofluor 10x objective for wider FOV, then, play around with it to see if you have a higher mag lens, such as 20x objective. 5) Refresh the DAB solution every 10-15 minutes. Tip: Background can be reduced by pre-treatment with 0.3% H2O2 in PBS. After photoconversion, the tissue can be osmicated and used for EM, but additional step might be necessary, such as gold-toning EM protocol, which can be found on the web.