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: Wash 3 times with 50 mM ammonium chloride

Step 2: Incubate for 5 mins with 50 mM ammonium chloride

Step 3: Incubate for 10 mins with 0.1% saponin in PBS

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

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

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

Axol’s Human iPSC-Derived Cortical Neurons express the synaptic marker VGLUT1 (green), TUJ1 (red) and DAPI (blue).