Ab134078

Cells were seeded at 10,000 cells/cm² in 8-well chamber slides and fixed with 4% freshly-prepared formaldehyde (pH 6.9) for 15 min at room temperature (RT) as previously done [58 (link)]. After fixation, the cells were permeabilized with 0.2% Triton-X100 in phosphate buffered saline (PBS) for 15 min at RT, followed by blocking with 1% BSA, glycine and 0.1% Tween-20 in PBS for 2 h at RT. Then cells were incubated with primary antibody at 1:250 dilution at 4 °C for 12 h, washed four times with 0.1% Tween-20 containing PBS (PBST) and incubated with secondary antibody (1:1000 dilution) at RT for 1 h. Finally, the slides were washed four times with PBST and the samples were mounted with DAPI-containing Fluoroshield mounting medium (#ab104139, Abcam, Cambridge, UK). Rabbit anti-human GHR monoclonal antibody (Abcam #ab134078) and goat anti-rabbit secondary antibody with AlexaFluor488 tag (#R37116, Life Technologies, Carlsbad, CA, USA) were used. Microscopic imaging was done using Cytation 3 imaging reader (BioTek, Winooski, VT, USA) and Gen5 2.09 software. The scale bar represents 100 μm. Magnification was at 20×.

Cells were seeded at 10,000 cells/cm² in 8-well chamber slides and transfection was performed as described above. Transfection media was replaced with antibiotic containing complete growth media after 24 hr and cells were fixed after 36 hr more (a total of 60 hr post-transfection), and cells were fixed with 4% freshly-prepared formaldehyde (pH6.9) / 15 min / 25C (using 100% methanol for fixation gave equally good results). Cells were permeabilized with 0.2% Triton-X100 in 1X PBS / 15 min / 25C, followed by blocking with 1% BSA / 4 hr / 25C. Incubation time was 12hr / 4C for primary antibody and 2 hr / 25C for secondary antibody. Finally, the slides were washed four times with 1X PBS and the sample was mounted with Fluoroshield mounting medium containing DAPI (Abcam #ab104139, Cambridge, UK), covered with a 60 mm coverslip and the edges were sealed with nail-polish and stored at 4C for microscopy. Microscopic imaging was done using a Nikon Eclipse E600 compound fluorescent microscope fitted with a Nikon DS-Fi1CC camera (Nikon, Tokyo, Japan) and NIS-Elements BR3.2 imaging software. Sera used were rabbit anti-human-Ki67 monoclonal antibody with AlexaFluor488 tag (Abcam #ab154201, 1:300 dilutions); rabbit anti-human GHR monoclonal antibody (Abcam #ab134078, 1:250 dilution); goat anti-rabbit secondary antibody with AlexaFluor488 tag (Life Technologies #R37116, 1:500 dilution).