Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs)

Prepare glass coverslips and plates for replating

Add 10 or 100 μg/mL of Poly-D-lysine onto plastic wells and glass coverslips, respectively and incubate at 37⁰C overnight.

Wash plenty with Phosphate-buffered Saline (PBS), at least 3 times.

Add Matrigel and incubate at 37⁰C for at least 1 hour.

Prepare media for thawing and replating

Pre-warm spinning falcons containing 9 mL of Neurobasal.

Prepare Day 16 (D16) media + ROCKi (1:1000).

Allow an aliquot of desired volume to reach room temperature.

Thawing and replating of Day 16 Medium Spiny Neurons (MSNs) precursors

Thaw cryovial containing Day 16 MSN precursors in water bath until only a small component remains frozen.

Carefully transfer contents of cryovial to pre-warmed spinning tubes.

Centrifuge at 350g for 5 min.

While spinning, aspirate Matrigel and replace with D16 media + ROCKi (1:1000).

Aspirate media from cell pellet in spinning falcon and replace with D16 media + ROCKi (1:1000), slowly and gently resuspending the pellet.

Transfer an appropriate amount of cell suspension into previously prepared glass coverslips and swirl plate gently in a figure 8 motion.

Allow cells to be cultured on glass coverslips as described in step 5 of Protocol: Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs) until relevant experimental timepoints for immunocytochemistry.

Transfer glass coverslips cultured with MSNs matured to relevant experimental timepoints into a 6-well plate.

Draw a fitting circle enclosing the entire coverslip with a hydrophobic Pap pen.

Fix cells by adding 2% PFA in PBS to each well at room temperature for 20 mins.

Remove PFA completely using a pipette.

Wash thoroughly 3 times with PBS, at least 10 mins each.

Incubate the coverslips in PBS for at least 1 hour at room temperature or at 4°C overnight.

Perform antigen retrieval by adding 50 mL of 1x citrate buffer (pH 6.0) to each well, and place the 6-well plate in a water bath at 80ºC for 5 mins.

Leave to incubate at room temperature for 10-20 mins.

Prepare blocking solution containing PBS with 10% Donkey Bovine Serum (NBS) and 0.1% Triton-X during the post-antigen retrieval incubation.

Remove the citrate buffer and add 50 mL of blocking solution.

Leave to incubate at room temperature for 10 mins.

Wash twice with PBS, at least 10 mins each.

During the previous washes, prepare the Primary Antibody Solution containing PBS with 10% Donkey Bovine Serum (NBS), supplemented with appropriate primary antibodies in their respective working concentrations.

The following primary antibodies (working concentration 1:250) were used in Do, Q. et al. (2023) for immunostaining: anti-CTIP2, anti-DARPP32, anti-DARPP32, GAD67, anti-MAP2, anti-Calbindin, PDYN, PKEN, Substance P, Tyrosine hydroxylase.

Remove all the PBS from each well and add 80 mL of the Primary Antibody Solution.

Leave to incubate overnight at 4°C.

Remove all the Primary Antibody Solution and add fresh PBS.

Wash thoroughly 3 times with PBS, at least 10 mins each.

During the last wash, prepare Secondary Antibody Solution containing PBS with 10% Donkey Bovine Serum (NBS), supplemented with appropriate secondary antibodies and DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) in their respective working concentrations.

The following secondary antibodies (working concentration 1:1000) were used in Do, Q. et al. (2023) for immunostaining: Alexa-Fluor 555 Mouse and Rabbit, Alexa-Fluor 648 Rabbit, Alexa Fluor 488 Rat and Chicken, and DAPI.

Remove all the PBS from each well and add 80 mL of the Secondary Antibody Solution.

Leave to incubate at room temperature for 1-2 hours.

Once done, wash well with PBS.

Wrap culture plates with aluminium foil and/or keep away from direct light.

Keep immunostained well plates in PBS plus 0.02% Azide for long-term storage.

Keep immunostained coverslips in PBS till 24 hours prior to imaging date.

At least 24 hours prior to the recording, apply a small drop of SlowFade Diamond Antifade mountant onto each coverslip, and carefully place the coverslip on the glass slides.

Leave to incubate the mounted slides at 4°C overnight.