9661, 1:100 in 1% phosphate-buffered saline with bovine serum albumin], and anti–Ki-67 probes (Dako (Glostrup, Denmark); check details 1:100). For evaluation of the amount of lipids, frozen sections were mounted on glass slides and stained with Oil Red O (Sigma-Aldrich, St. Louis, MO). All histopathology was evaluated by an experienced pathologist

in a blinded study setting. The pathology findings were used to cross-validate the longitudinal changes in the optical end-points. Intrinsic fluorescence in tumor was imaged using a two-photon confocal microscopy setup. These experiments were carried out to relate the differences in fluorescence spectra obtained with AFS to specific structures in the tissue slices. Snap-frozen tumor pieces were sliced in thick sections (25 μm), kept unstained and unfixed, and mounted onto glass microscope slides. The two-photon learn more excitation source was a Ti:Sapphire laser (Tsunami, Spectra Physics, Santa Clara, CA) tuned to 790 nm. The excitation light (equivalent to a single-photon excitation wavelength of 395 nm) was delivered to, and the emitted light was collected from the sample through a Leica Confocal microscope [with a Leica (Mannheim, Germany) HCX

IRAPO 25 × water immersion objective with an NA of 0.95] coupled to a Leica TCS SP5 tandem scan head operating at 500 lines per second. A photomultiplier served as the detector. For each tumor sample, fluorescence images were obtained in the wavelength ranges of 400 to 500 nm, 500 to 600 nm, and 600 to 700 nm. This was done to compare the relative intensity of fluorescence at these spectral ranges between treated and control animals. To examine the trends in optical parameters over time, a linear regression model was performed in MATLAB 7.13 (MathWorks Inc, Natick, MA). The fixed-effects terms in the models were treatment (controls vs cisplatin), time (day), and their

interactions. Rapamycin chemical structure A slope and intercept were fit for the data of both the treated and control groups using maximum likelihood estimation. For the significance of fixed effects, a likelihood ratio test was statistically compared to a χ2 distribution with 1 df (for one coefficient being eliminated). For all tests, statistical significance was set at P < .05. DRS parameter quantification was performed as part of the model-based data analysis using a total of 712 DRS spectra. The longitudinal changes for the average tumor volume and various DRS parameters over time are shown in Figure 2. In the control animals, the tumor volume increased during the entire follow-up period, whereas the tumors of the cisplatin-treated animals started to shrink 2 days after treatment. For the DRS parameters, the trends during follow-up were significantly different between the treated and the control groups for the Mie-scattering slope (P < .0001), Mie-to-total scattering fraction (P < .001), tissue oxygenation (P = .035), and fat volume fraction (P < .0001).