Product has been added to your cart.
Contact
  1. Home
  3. Resources
  5. How To Guides
  7. How to Videos and Guides
  9. How to Perform Immunocytochemistry (ICC)

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-Bioscience-human-iPSC-image-ir_attachment_78526-1.png

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).