Anti-Ki-67 antibody ValidAb^TM

HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SPE confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective and 405nm (24.5% power, PMT: 688V gain) and 488nm (29.0% power, PMT: 775V gain) lasers. Images were captured as a stack (0.23µm z-spacing) before being deconvolved with Huygens professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Serum starvation inhibits the ability of HEK293T cells to proliferate therefore inhibits Ki-67 expression. Through stimulation with 20% FBS this then strongly promotes proliferation therefore driving high expression of Ki-67. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Following this all coverslips were then fixed with ice-cold 4% paraformaldehyde in PBS. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 550 (Thermofisher, 84540) conjugated secondary antibody (1:300 dilution) and FITC Phalloidin (HB0814, 1:500 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a DFC365FX monochrome digital camera. All images were captured using a 40x objective and the same exposure settings of 73.9ms (DAPI), 73ms (FITC Phalloidin) and 107.2ms (Dy550). Images were captured as a stack (0.53µm z-spacing) before being deconvolved in Huygens Professional and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7502 produces reliable Ki-67 staining at concentrations as low as 1:4,000 (0.25µg/ml). Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at concentrations ranging from 2µg/ml (1:500 dilution) to 0.25µg/ml (1:4,000 dilution). This was followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. Images were captured as a z-stack using a 100x objective with the following exposures:

• 1:500 – DAPI: 4.9ms, L5: 20ms
• 1:1,000 – DAPI: 4.9ms, L5: 10ms
• 1:2,000 – DAPI: 7.9ms, L5: 61ms
• 1:4,000 – DAPI: 6ms, L5: 15.2ms

Images were deconvolved using Huygens Professional before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7052 reveals the expression of Ki-67 in proliferating HEK293T cells in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (1.1% power, HyD: 10% gain). Images were captured as a stack (0.38µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7052 reveals proliferating cells within a large population of HEK293T cells grown in culture. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:500 dilution (2µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B monochrome digital camera. The image was captured as a tilescan and z-stack (0.3µm spacing) using a 40x objective, DAPI (4.9ms exposure) and L5 (9.1ms exposure) filters. Images were combined in LASX, deconvolved in Huygens professional then flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Serum starved cells (SS) express only degradation products of Ki-67 whereas 20% FBS stimulation (SS + Stim) induces new Ki-67 expression as seen by the presence of higher molecular weight bands. Method: Following splitting, HEK293T cells were cultured on PLL coated coverslips for 24 hours in 10% FBS in DMEM with 1% Pen/Strep. Cells were washed with plain DMEM and then incubated with DMEM (1% Pen/Strep) for around 20 hours before half of coverslips were then stimulated with 20% FBS for 2hr 45m. Cells were subsequently lysed in Laemmli sample buffer. Rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Samples were loaded (20µg / lane) onto a 4-20% acrylamide gel alongside a protein ladder (NEB Prestained) before being run at 150V for 50 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7052 at a 1:500 dilution (2µg/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in a 1:10,000 dilution of a polyclonal goat anti-mouse HRP conjugated secondary antibody (Sigma Aldrich A3682) for 2hrs and visualized again using Hello Bio ECL and a Licor Odyssey Fc imaging system (ECL channel: 4.5 min exposure, 700nm channel: 30 sec exposure).

HB7052 reveals the subcellular expression of Ki-67 in the nucleus of a proliferating HEK293T cell. Method: HEK293T cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7052 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse DyLight 488 (Thermofisher, 35503) conjugated secondary antibody (1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (8.6% power, PMT: 800V gain) and 488nm (3.1% power, HyD: 52.4% gain). Images were captured as a stack (0.12µm z-spacing) before being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).