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How to Perform Immunocytochemistry (ICC)

Sample Preparation and Fixation

Step 1: Add cell culture-grade coverslips to wells

Step 2: Make 1X solution of Axol Sure BondTM from the 50X stock using PBS, e.g. 240 μL in 12 mL PBS

Step 3: Add enough 1X Axol Sure BondTM to each well to immerse the coverslips and incubate overnight at 37oC

Step 4: Wash coverslips with sterile H20 3 x 5 mins

Step 5: Grow cells on coated coverslips for desired length of time

Step 6: When ready to stain cells, rinse them briefly in PBS

Step 7: Fix the samples using 4% paraformaldehyde in PBS pH 7.4 for 15 minutes at room temperature

Step 8: Wash the samples twice with PBS

Cell Permeabilization and Blocking

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

Step 2: Wash cells in PBS 3 x 5 mins

Step 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 antibody was raised diluted in PBS e.g. 2.5 mL serum in 47.5 mL PBS)

Staining

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

Step 2: Incubate the cells in the primary antibody solution in a humidified chamber for 1 hr at room temperature or overnight at 4oC

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

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

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

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

Mounting and Counter-Staining

Step 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

Step 2: Seal the edges of the coverslip with nail polish

Step 3: Store in the dark at 4oC

Top Tips:

1. For fixation and incubation steps use a rocker to ensure even distribution of fixative/antibody solutions

2. For intracellular target proteins, cell permeabilization is essential

3. Triton X:100 disrupts membranes so do not use this with membrane-associated targets

The following list includes the primary antibodies that can be used for characterizing neurons and astrocytes derived from Axol iPSC-derived NSCs:

Tuj1 - Neuronal Marker (axon)

MAP2 - Neuronal Marker (dendrite), Novus NB300-213

Doublecortin - Newborn Neuron

Tbr1 Deep-layer Cortical Neurons

Brn2 - Upper-layer Cortical Neurons

VGlut1 - Glutamatergic Neurons

S100 - Astrocytes

PSD-95 - Postsynaptic Terminals

Synaptophysin - Presynaptic Terminals

Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes after 8 days of culture
Axol’s Human iPSC-derived Striatal neurons at day 18 and day 33 in culture express GABA (red, day 18), Calbindin (red, day 33), DARPP32 (red, day 33) and MAP2 (green, day 18 & 33). Counterstained with DAPI (blue).