Faint fluorescent signal was detected in the positive control cells. No signal was detected in the negative control cells or isotype negative control.
Legend: Immunofluorescence. Nestin (green fluorescence) is present in positive control cells (PC3) and absent in negative control cells( MCF7). Scale bar = 50 μm, Magnification = 20X, FITC Exposure = 1.5s, Insets at 40X magnification.
Left panel: Micrograph image of PC3 Nestin-positive prostate adenocarcinoma cells (top) and MCF7 Nestin-negative breast adenocarcinoma cells (bottom) with 15 μg/mL of anti-Nestin-FITC Ab.
Middle panel: Micrograph image of PC3 Nestin-positive prostate adenocarcinoma cells (top) and MCF7 Nestin-negative breast adenocarcinoma cells (bottom) with 10 μg/mL of anti-Nestin-FITC Ab.
Right panel: Micrograph image of reagent isotype control FITC-conjugated mouse IgG1 antibody on PC3 Nestin-positive prostate adenocarcinoma cells (top) and MCF7 breast adenocarcinoma cells (bottom).
Antigen: Nestin (NES) (FITC)
Catalog number: ABIN1774766
Lot number: I030314
Dilution: 1:75, 1:100
Prostate and breast adenocarcinoma cell lines were grown directly on chamber slides, washed with 1X PBS, and fixed with 4% paraformaldehyde in 1X PBS for 15 min at room temperature (RT).
Fixed cells were rinsed three times in PBS for 5 min each at RT.
Cells were blocked in 1X PBS / 5% normal goat serum / 0.3% Triton X-100 for 60 min at RT.
Cells were rinsed two times in 1X PBS.
Cells were incubated with primary antibody diluted 1:75 and 1:100 in 1X PBS / 1% BSA / 0.3% Triton X-100 overnight at 4°C in dark conditions.
Cells were rinsed three times in PBS for 5 min each at RT.
Coverslips were mounted on slides with DAPI-Fluoromount-G.
Stained cells were imaged with a Nikon Eclipse E600 microscope and Olympus DP70 camera.
The anti-Nestin antibody showed positive staining of weak intensity localized appropriately to the cell cytoplasm in a pattern consistent with intermediate filaments in positive control PC3 cells. No staining was seen in the negative control MCF7 cells or with the isotype control antibody. Although specificity of the antibody was acceptable, its sensitivity with the current protocol was questionable. It is, however, entirely possible that if this antibody is used with an appropriate amplification protocol the sensitivity would be increased.
Purified IgG conjugated to Fluoroscein using Innova Biosciences Lightning-Link®, supplied in Phosphate buffered saline (PBS) containing 0.09 % Sodium azide
Precaution of Use
This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.