Subcutaneous tumors, produced by intradermal injection of human A431 cells, were preconditioned with intramuscular MTX or with saline for 1 d or 3 d, and then PpIX production was initiated by intraperitoneal ALA injection as described in Methods. (A) Harvested tumors were cryosectioned and analyzed by PpIX-specific confocal microscopy (a, c) and by post-fixation and H&E staining to visualize the corresponding region (b, d). Bright PpIX signal (asterisk) was observed preferentially in MTX-treated tumors, which despite some areas of necrosis, had high PpIX levels in living tumor areas. (B) Quantitation of PpIX signal in digital confocal images, as a function of MTX pre-conditioning for 1 day (1x MTX) or for 3 days (3x MTX). Values are mean ± SD; the number of fields and number of mice (in parentheses) examined are shown above each bar.