Alexa fluor 647 mouse anti ki 67 antibody

Cells were trypsinized and harvested in phenol-red free medium supplemented with FBS. Cells (1 × 10⁶) were washed and suspended in 500 ul of phosphate-buffered saline (PBS). The cells were fixed with ice-cold 100% ethanol and incubated at −20 °C overnight. The cells were pelleted and washed 2X with PBS and incubated with Alexa Fluor® 647 Mouse anti-Ki-67 antibody (Catalog No.558615, BD Biosciences) for 30 minutes at room temperature. Cells were then washed 2X with PBS and suspended in 500 ul of propidium iodide/RNase solution and incubated in the dark at room temperature for 4 hours. Cell-cycle distribution and Ki67 expression were determined using the BD LSR II analyzer (BD Biosciences) at the Microscopy, Image and Cytometry Resources Core at Karmanos Cancer Institute. The data were analyzed using ModFit software.

Tumor tissue sections were washed 3x with PBS-T (PBS, 0.1% Tween) and briefly air-dried. Using a hydrophobic pen (Dako, Angilent), a circle was drawn around the tumor sections, and they were then incubated with 1% Triton X- at room temperature for 10 min in the dark. After a new wash with PBS-T, the tissue sections were incubated with 2% bovine serum albumin (BSA) at room temperature for 30 min in the dark. The Alexa Fluor 647 Mouse anti-Ki-67 antibody (BD Biosciences, #558615) was used at 1:50 dilution in BSA at room temperature for 1.5 hr in the dark. For Hypoxyprobe-1 staining, slides were incubated overnight at 4°C with the primary antibody (dilution at 1:50). On the following day, mouse (#A21237) secondary Alexa Fluor 647(Invitrogen) antibody was used at 1:1000 dilution for 1.5 hr at RT. New PBS-T wash was followed by staining with DAPI at 1:1000 dilution in PBS at room temperature for 15 min in the dark. Slides were washed and mounted with anti-fade solution. Images were taken with an Olympus (UPLFLN 10×) objective using Cytation 5 microscope (BioTek Instruments). Image analysis was performed by determining the ratio of Ki67+ nuclei over total nuclei in each RFP and GFP channels using the image calculator tool available on ImageJ.