An issue which recurs is cytospin prepared slides where the required information is localization of proteins or other lebaled molecules. The problem is that cytospin so smashes the cells that this is not possible.

Cells may be adhered to coverslips with poly-L-lysine to preserve morphology.


This protocol is from the Microscopy Core at NYU Langone. I made very minor changes to it.:

For LM:
1. Poly-L-Lysine Stock: (Sigma, P1274, MW 70,000-150,000Da)
            Dissolve in bidistilled water at 2mg/ml
            Or: order 0.1% stock solution (Sigma P8920), Aliquots can be stored at -20°C

2. Coverslips: most typically #1.5.

3. Coating of coverslips to mount sections for light microscopy
            a. Clean round glass coverslips with either ethanol, or 1% hydrochloric acid in 70% ethanol, or with ammonia (no residue on coverslip).
            b. Rinse with bidistilled water.
            c. Dry coverslips or slides separately on a filter paper in a large dish.
            d. Prepare a solution containing 0.2mg/ml (W/V) poly-L-Lysine in bidistilled water or use the commercially available 1% stock solution. Place a 30 to 50 ul drop in the center of the coverslip with the solution.
            e. Allow the coating to settle for at least 30 min in a moist chamber.
            f. Rinse briefly with bidistilled water and dry face up on a filter paper for 1 h at 60°C or overnight at ambient temperature.
            g. Coverslips can be coated in large batches and stored indefinitely in dust-free dishes at room temperature.
            h. UV treat the coverslip for live cell cultures before use it.

4. Immunofluorescent staining
            a. Fix the cells with 2-4% PFA in PBS for 5 – 10 minutes;
            b. Put 50 to 100 uL cells in the center of coverslip and let it settled for 20 minutes in a moist chamber, then continue IHC staining procedure.


Other excellent protocols here.


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