Confocal immunofluorescent analysis of MCF7 cells using EpCAM (D1B3) Rabbit mAb #2626 (green) either untreated (left), or treated with Image-iT® FX Signal Enhancer (right). Note the reduction in non-specific, nucleolar background following pre-treatment with Image-iT® FX Signal Enhancer.
Following specimen preparation:
1. Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
2. Rinse three times in PBS for 5 minutes each.
3. Apply 3–4 drops of Image-iT™ FX Signal Enhancer (or sufficient volume to cover the cells) and incubate for 30 minutes at room temperature.
4. Rinse three times with PBS for 5 minutes each.
5. Block specimen in Blocking Buffer for 60 minutes.
6. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
7. Aspirate blocking solution, apply diluted primary antibody.
8. Incubate overnight at 4°C.
9. Rinse three times in PBS for 5 minutes each.
10. Coverslip slides with Prolong® Gold Antifade Reagent.
11. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
Store at room temperature. This product is stable for 12 months.
Alexa Fluor® and many other anionic fluorescent dyes and proteins can bind nonspecifically with cationic cell and tissue constituents. By efficiently blocking these nonspecific electrostatic interactions, Image-iT® FX Signal Enhancer can dramatically improve the signal-to-noise ratio of immunolabeled cells and tissues. Image-iT® is a liquid that is applied directly to slides or coverslips containing fixed and permeabilized cell or tissue samples prior to staining with fluorescent probes.
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