Axol Guide to Performing Immunocytochemistry

Learn the immunocytochemistry methods for staining  neural progenitors and neurons, including a special protocol for synaptic markers!

General Immunocytochemistry Protocol

Sample Preparation and Fixation

1. Add cell culture-grade coverslips to wells.

2. Add enough Axol ReadySet to immerse the coverslips.

3. Incubate at 37ºC for 45 minutes.

4. During the incubation period, make 1X solution of  Axol Sure Bond  from the 50X stock using PBS, e.g. 240μL in 12 mL PBS.

5. Wash the plate 4 times using an equal volume of ddH2O e.g. if 250 μL Axol ReadySet, use 250 μL ddH2O per wash.


Warning: Axol ReadySet must not be allowed to dry out following the wash step. Proceed straight to coating with Axol Sure Bond.
 

6. Add enough 1X Axol Sure Bond to each well to immerse the coverslips and incubate for 1 hour at 37°C.

7. Remove Axol Sure Bond from the coverslips.

Warning: Do not wash the cover slips after coating with  Axol Sure Bond .


7. Grow cells on coated coverslips for desired length of time.

8. When ready to stain cells, rinse them briefly in PBS.

9 Fix the samples using 4% paraformaldehyde in PBS pH 7.4 for 15 minutes at room temperature.

10. Wash the samples twice with PBS.

Cell Permeabilization & Blocking

1. To permeabilize the cells incubate the samples for 10 mins in PBS containing 0.3% Triton X-100.

2. Wash cells in PBS 3 x 5 mins.

3. To prevent non-specific antibody binding, incubate the samples for 1 hr with blocking buffer (5% serum from the species in which the secondary was raised diluted in PBS e.g. 2.5 mL serum in 47.5 mL PBS).

Staining

1. Dilute the primary antibody in blocking buffer using the dilution factor recommended by the antibody datasheet guidelines.

2. Incubate the cells in the primary antibody solution in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

3. Dilute the secondary antibody in blocking buffer using the dilution factor recommended by the antibody guidelines.

4. Remove the primary antibody solution and then wash cells 3 x 5 mins with PBS.

5. Incubate the cells in the secondary antibody solution for 1 hr at room temperature in the dark.

6. Remove the secondary antibody and then again wash cells with PBS 3 x 5 mins in the dark.

Mounting and Counter-Staining

1. Mount stained coverslip on slides using a drop of mounting medium containing DAPI (to counter stain the cell nucleus) according to manufacturer’s guidelines e.g. ProLong Gold Antifade Reagent, Life Technologies.

2. Seal the edges of the coverslip with nail polish.

3. Store in the dark at 4°C.

Synaptic Marker ICC Protocol

Sample Preparation and Fixation

1. Add cell culture-grade coverslips to wells.

2. Add enough Axol ReadySetto immerse the coverslips.

3.   Incubate at 37ºC for 45 minutes.

4. Make 1X solution of  Axol Sure Bond  from the 50X stock using PBS, e.g. 240μL in 12 mL PBS.

5. Wash the plate 4 times using an equal volume of ddH2O e.g. if 250 μL Axol ReadySet, use 250 μL ddH2O per wash.


Warning: Axol ReadySet must not be allowed to dry out following the wash step. Proceed straight to coating with Axol Sure Bond.
 

6. Add enough 1X Axol Sure Bond to each well to immerse the coverslips and incubate for 1 hour at 37°C.

7. Remove 
Axol Sure Bond from the coverslips.
 

Warning:  Do not wash the cover slips after coating with  Axol Sure Bond .


8. Grow cells on coated coverslips for desired length of time.

9. When ready to stain cells, rinse them briefly in PBS.

10. Fix the samples using 4% paraformaldehyde in PBS pH 7.4 for 15 minutes at room temperature.

11. Wash the samples twice with PBS.

Cell Permeabilization & Blocking

1. Wash 3 times with 50 mM ammonium chloride.

2. Incubate for 5 mins with 50 mM ammonium chloride.

3. Incubate for 10 mins with 0.1% saponin in PBS.

4. Incubate for 30 mins in blocking buffer (PBS containing 3% BSA & 0.1% saponin).

Staining

1. Dilute primary antibody in blocking buffer using manufacturer’s recommended dilution.

2. Put a piece of parafilm on wet Whatman paper and apply 200 μL of primary antibody solution to the top of the parafilm.

3. Put coverslips upside down on primary antibody solution. Incubate for 1 hr at room temperature.

4. Transfer coverslips back to a tissue culture plate e.g. 12 well plate.

5. Wash twice with 0.1% saponin in PBS.

6. Incubate for 10 mins with blocking buffer.

7. While samples are in blocking, dilute secondary antibody in blocking buffer using manufacturer’s recommended dilution.

8. Put a new piece of parafilm on wet Whatman paper and apply 200 μL of secondary antibody solution.

9. Put coverslips upside down on secondary antibody solution, as before so that cells are in contact with solution. Incubate for 1hr at room temperature.

10. Transfer coverslips to your tissue culture plate e.g. 12 well plate.

11. Wash twice with 0.1% saponin in PBS.

12. Wash twice with PBS.

Mounting and Counter-Staining

1. Mount stained coverslip on slides using a drop of mounting medium containing DAPI (to counter stain the cell nucleus) according to manufacturer’s guidelines e.g. ProLong Gold Antifade Reagent, Life Technologies.

2. Seal the edges of the coverslip with nail polish.

3. Store in the dark at 4°C.

Get the PDF version of the protocol for use in the lab!

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