Mouse anti human ki 67 antibody

A549 Cells were cultured in 96-well plates, at 8,000 cells/well in respective growth media. Following overnight incubation media were replaced with media containing 0.5% FBS and Cannabis extracts were added in sub-lethal concentrations, as determined in preliminary experiments (1-2 µg/ml). Extract were then incubated for 48 h in 37 °C with 5% CO₂. Following this, cells were fixed using 4% paraformaldehyde, permeabilized using 0.1% Triton and blocked using 5% Normal Donkey Serum (Jackson ImmunoResearch Inc., 017-000-121) in PBS. Following blocking, cells were incubated with mouse anti-human Ki-67 antibody (BD Biosciences, 610969) overnight at 4°C. This was followed by incubation with Alexa Fluor® 488-conjugated donkey anti-mouse antibody (Thermo Fisher Scientific, A-21202) and counterstained with Hoechst (2 µg/ml). Cells were visualized with ImageXpress® Micro System at 10× magnification. The percentage of proliferation was calculated as the number of Ki-67 positive cells divided by the number of total cells counted in each well, multiplied by 100.

The distal colon sections were deparaffinized in xylene and rehydrated. The sections were then heated in 0.1mM sodium citrate buffer (pH 6.2) for antigen retrieval. Samples were incubated with mouse anti-human Ki-67 antibody (BD Biosciences, San Diego, CA), biotinylated rabbit anti-mouse antibody (Agilent, Santa Clara, CA) and Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) [33 ]. Samples were stained with DAB (3,3′-diaminobenzidine) then counter-stained with Harris Hematoxylin. Samples were dehydrated and mounted, and the numbers and locations of proliferative cells were recorded. Observers were blind to the treatment and were only given a number for each animal as a reference. For each animal, the number and position of each labelled cell in the crypt column were recorded. There were at least 15 fields examined per animal, then averaged. To calculate proliferation index (PI), the number of proliferative cells was divided by the number of total cells per crypt, then multiplied by 100. To calculate proliferative zone (PZ), the position of the uppermost labeled cell was divided by the total number of crypt cells, then multiplied by 100.