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
