TITLE: Immunostaining of corticostriatal culture on fluid-walled dumbbells
AUTHORS: Quyen Do, Federico Nebuloni, Richard Wade-Martins

[DESCRIPTION]
This protocol describes the immunocytochemistry of the iPSC-derived corticostriatal culture on fluid-walled dumbbells. 

[BEFORE_START]
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: Generation of the corticostriatal pathway coculture on fluid-walled dumbbells
1. Fabricate the fluid-walled dumbbells on 6 cm culture dishes as described in step 1 of Protocol: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
SECTION: Generation of the corticostriatal pathway coculture on fluid-walled dumbbells
2. Allow cells to be cultured on glass coverslips as described in Section: Establishment of Human Corticostriatal Pathway in Protocol: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway until relevant experimental timepoints for immunocytochemistry.
SECTION: PFA Fixation
3. At day 25 of the coculture, remove all cell media and FC40 by gentle pouring. 

SECTION: PFA Fixation
4. Wash the whole dish with PBS twice using P1000.
SECTION: PFA Fixation
5. Fix cells by adding 2% PFA in PBS to each dish at room temperature for 20 mins.
SECTION: PFA Fixation
6. Remove PFA completely using a pipette.
SECTION: PFA Fixation
7. Wash thoroughly 3 times with PBS, at least 10 mins each.
SECTION: PFA Fixation
8. Incubate the coverslips in PBS for at least 1 hour at room temperature or at 4°C overnight.
SECTION: Heat-induced Antigen Retrieval
9. Perform antigen retrieval by adding 2 mL of 1x citrate buffer (pH 6.0) to each dish, and place the culture dish in a water bath at 80ºC for 5 mins.
 

SECTION: Heat-induced Antigen Retrieval
10. Leave to incubate at room temperature for 10-20 mins.
SECTION: Heat-induced Antigen Retrieval
11. 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
12. Remove the citrate buffer and add 1 mL of blocking solution.
SECTION: Heat-induced Antigen Retrieval
13. Leave to incubate at room temperature for 10 mins.
SECTION: Heat-induced Antigen Retrieval
14. Wash twice with PBS, at least 10 mins each.
SECTION: Primary Antibody Incubation
15. During 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-DARPP32, anti-DARPP32, anti-MAP2, anti-GFP.
SECTION: Primary Antibody Incubation
16. Remove all the PBS from each well and add 80 mL of the Primary Antibody Solution.
SECTION: Primary Antibody Incubation
17. Leave to incubate overnight at 4°C.
SECTION: Secondary Antibody Incubation
18. Remove all the Primary Antibody Solution and add fresh PBS.
SECTION: Secondary Antibody Incubation
19. Wash thoroughly 3 times with PBS, at least 10 mins each.
SECTION: Secondary Antibody Incubation
20. 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, Alexa-Fluor 648 Rabbit, Alexa Fluor 488 Chicken, and DAPI.
 

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

SECTION: Secondary Antibody Incubation
23. Once done, wash well with PBS.
SECTION: Secondary Antibody Incubation
24. Incubate the culture in generous amount of PBS Azide until imaging.