Alexa fluor 488 anti rat secondary antibody

To determine extent of apoptosis induced by different formulations, a TUNEL assay kit (Promega) was used. Briefly, paraffin-embedded sections were deparaffinized with xylene and rehydrated using an ethanol gradient. Sections were incubated with the TUNEL enzyme mixture for 1 h following the manufacturer’s instructions and nuclei were stained using Prolong Gold Antifade Mountant with DAPI. The sections were imaged using an LSM710 confocal microscope (data collection: Zen 2012 SP5, Zeiss; data processing: Fiji-ImageJ version 1.52p).
Expression of proliferation marker ki67 was monitored using immunohistochemistry. Rehydrated tumor sections were immersed in sodium citrate buffer (pH 6.0) and heated at 95 °C for 20 min to retrieve antigen. Sections were blocked using goat serum (10% in Tris Buffered Saline), incubated with rat anti-mouse Ki67 (Clone SolA15) antibody (eBiosciences, catalog # 14-5698-82, 1:100 in blocking buffer) for 1 h followed by a 30-min incubation with Alexa-Fluor 488 anti-rat secondary antibody (Invitrogen catalog # A-11006, 1:500 in Tris Buffered Saline). Nuclei were stained using Prolong Gold Antifade Mountant with DAPI. The sections were imaged using an LSM710 confocal microscope (data collection: Zen 2012 SP5, Zeiss) and the nuclei were pseudo-colored to red during image processing using Fiji-ImageJ version 1.52p.

After the last DCE-MRI imaging scan, one animal from each group was sacrificed and the tumors were harvested for CD31 analysis. Tumors were imbedded in optimal-cutting-temperature (O.C.T.) compound and later on sectioned into 4-µm thick slices. Tumor sections were fixed with ice-cold acetone for 20 min. After blocking non-specific binding sites with 1% BSA for 30 min, the sections were incubated with anti-CD31 antibody (Abcam) for 1 h at room temperature. After PBS wash, the sections were incubated with Alexa Fluor 488 anti-rat secondary antibody (Invitrogen) at room temperature. The slides were then mounted with DAPI-containing mounting medium after PBS wash. Fluorescence images were taken on a fluorescence microscope (Leica, Germany). Quantitative analysis of CD31-positive area was performed by ImageJ software. For hematoxylin and eosin (H&E) staining, one tumor from each group was collected 5 days post PTT and fixed with 4% formaldehyde solutions at room temperature for 48 h.