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1. Wash cells gently with PBS at room temperature.
2. Fix with cold methanol (-20 °C) for 5 minutes. See Fixation for other options.
3. Fix in (-20 °C) acetone for 2 minutes.
4. Rinse 3 times with PBS.
5. Cover cells with 100 ml properly diluted primary antibody in PBS.
Cover with a coverslip. Incubate for 1 hour at 37 °C or room temperature.
6. Rinse 3X 10 minutes with PBS.
Immunofluorescence
7. Cover the cells with properly diluted secondary antibody couple to FITC or Texas-Red. Cover with a coverslip.
Incubate 0.5-2 hours at room temperature or 4 °C.
8. Wash with PBS, 3 x 10 minutes.
9. Mount and view under a fluorescence microscope.
Immunoperoxidase
7a. Cover the cells with properly diluted secondary antibody (biotinylated). Cover with a coverslip.
Incubate 0.5-2 hours at room temperature or 4 °C.
8a. Wash with PBS, 3 x 3 minutes.
9a. Apply Strepavidin/HRP and incubate at room temperature for 30 minutes.
10a. Wash with PBS, 3 x 3 minutes.
11a. Stain with DAB.
12a. Wash with running tap water for 3 minutes.
13a. Mount.
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