TITLE: Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs)
AUTHORS: Quyen  Do, Richard Wade-Martins

[DESCRIPTION]
This protocol describes the immunolabelling of one or several protein targets on PFA-fixed cell culture on glass coverslips. 

[GUIDELINES]
Adherent cells are prone to peeling, and hence, addition and removal of any liquid should be performed slowly with care.
 
At no point during the entire procedure prior to mounting should the sample be left to dry. 

Fluorescent-dye conjugated Secondary Antibodies are light-sensitive and hence should always be protected from light.

All PBS washes should be at least 10 mins, and plates are left to incubate on the bench at room temperature.

[STEPS]
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
1. Prepare glass coverslips and plates for replating
SECTION: PFA Fixation
4. Transfer glass coverslips cultured with MSNs matured to relevant experimental timepoints into a 6-well plate.
SECTION: PFA Fixation
5. Draw a fitting circle enclosing the entire coverslip with a hydrophobic Pap pen. 

 

 

SECTION: PFA Fixation
6. Fix cells by adding 2% PFA in PBS to each well at room temperature for 20 mins.
SECTION: PFA Fixation
7. Remove PFA completely using a pipette.
SECTION: PFA Fixation
8. Wash thoroughly 3 times with PBS, at least 10 mins each.
SECTION: PFA Fixation
9. Incubate the coverslips in PBS for at least 1 hour at room temperature or at 4°C overnight.
SECTION: Heat-induced antigen retrieval
10. 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.

 

SECTION: Heat-induced antigen retrieval
11. Leave to incubate at room temperature for 10-20 mins.
SECTION: Heat-induced antigen retrieval
12. Prepare blocking solution containing PBS with 10% Donkey Bovine Serum (NBS) and 0.1% Triton-X during the post-antigen retrieval incubation.
SECTION: Heat-induced antigen retrieval
13. Remove the citrate buffer and add 50 mL of blocking solution.
SECTION: Heat-induced antigen retrieval
14. Leave to incubate at room temperature for 10 mins.
SECTION: Heat-induced antigen retrieval
15. Wash twice with PBS, at least 10 mins each.
SECTION: Primary Antibody Incubation
16. 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.
SECTION: Primary Antibody Incubation
17. Remove all the PBS from each well and add 80 mL of the Primary Antibody Solution.
SECTION: Primary Antibody Incubation
18. Leave to incubate overnight at 4°C.
SECTION: Secondary Antibody Incubation
19. Remove all the Primary Antibody Solution and add fresh PBS.
SECTION: Secondary Antibody Incubation
20. Wash thoroughly 3 times with PBS, at least 10 mins each.
SECTION: Secondary Antibody Incubation
21. 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. 

 

SECTION: Secondary Antibody Incubation
22. Remove all the PBS from each well and add 80 mL of the Secondary Antibody Solution.
SECTION: Secondary Antibody Incubation
23. Leave to incubate at room temperature for 1-2 hours.

 

SECTION: Secondary Antibody Incubation
24. Once done, wash well with PBS.
SECTION: Mounting
25. Wrap culture plates with aluminium foil and/or keep away from direct light.
SECTION: Mounting
26. Keep immunostained well plates in PBS plus 0.02% Azide for long-term storage.

 

SECTION: Mounting
27. Keep immunostained coverslips in PBS till 24 hours prior to imaging date.

 

SECTION: Mounting
28. 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.
SECTION: Mounting
29. Leave to incubate the mounted slides at 4°C overnight.

 

SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
1.1. Add 10 or 100 μg/mL of Poly-D-lysine onto plastic wells and glass coverslips, respectively and incubate at 37⁰C overnight.
 

SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
1.2. Wash plenty with Phosphate-buffered Saline (PBS), at least 3 times.
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
1.3. Add Matrigel and incubate at 37⁰C for at least 1 hour.
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
2. Prepare media for thawing and replating
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
2.1. Pre-warm spinning falcons containing 9 mL of Neurobasal. 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
2.2. Prepare Day 16 (D16) media + ROCKi (1:1000). 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
2.3. Allow an aliquot of desired volume to reach room temperature. 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3. Thawing and replating of Day 16 Medium Spiny Neurons (MSNs) precursors
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.1. Thaw cryovial containing Day 16 MSN precursors in water bath until only a small component remains frozen.
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.2. Carefully transfer contents of cryovial to pre-warmed spinning tubes. 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.3. Centrifuge at 350g for 5 min.
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.4. While spinning, aspirate Matrigel and replace with D16 media + ROCKi (1:1000). 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.5. Aspirate media from cell pellet in spinning falcon and replace with D16 media + ROCKi (1:1000), slowly and gently resuspending the pellet. 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.6. Transfer an appropriate amount of cell suspension into previously prepared glass coverslips and swirl plate gently in a figure 8 motion. 
SECTION: Replating human Medium Spiny Neurons (MSNs) onto glass coverslips
3.7. 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.