Immunostaining of corticostriatal culture on fluid-walled dumbbells
Richard Wade-Martins, Quyen Do, Federico Nebuloni
Abstract
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
Generation of the corticostriatal pathway coculture on fluid-walled dumbbells
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
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 Protocol: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway until relevant experimental timepoints for immunocytochemistry.
PFA Fixation
At day 25 of the coculture, remove all cell media and FC40 by gentle pouring.
Wash the whole dish with PBS twice using P1000.
Fix cells by adding 2% PFA in PBS to each dish 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.
Heat-induced Antigen Retrieval
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.
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 1 mL of blocking solution.
Leave to incubate at room temperature for 10 mins.
Wash twice with PBS, at least 10 mins each.
Primary Antibody Incubation
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.
Remove all the PBS from each well and add 80 mL of the Primary Antibody Solution.
Leave to incubate overnight at 4°C.
Secondary Antibody Incubation
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, Alexa-Fluor 648 Rabbit, Alexa Fluor 488 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.
Incubate the culture in generous amount of PBS Azide until imaging.
