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How to Perform Immunocytochemistry (synaptic)

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)

Staining

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

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

Step 3: Put coverslips upside down on primary antibody solution

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

Step 5: Wash twice with 0.1% saponin in PBS

Step 6: Incubate for 10 mins with blocking buffer

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

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

Step 9: Put coverslips upside down on secondary antibody solution, as before so that cells are in contact with solution

Step 10: Incubate for 1hr at room temperature

Step 11: Transfer coverslips to your tissue culture plate e.g. 12 well plate

Step 12: Wash twice with 0.1% saponin in PBS

Step 13: Wash twice with PBS

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

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