• ioGABAergic Neurons (io1003) cultured in a 24 well plate following the user manual to day 12 at a seeding density of 150,000 cells/cm²

• Biological safety cabinet with a carbon filter (MSC-CF)

• Normoxic cell culture incubator (37°C, 5% CO²)

• -80°C freezer

• 1000 μL, 200 μL, 20 μL and 10 μL pipettes

• Standard light microscope

• Epifluorescent microscope

• 1.5 mL microcentrifuge tubes (Starlab, S1615 5510)

• 15 mL centrifuge tube, conical (Greiner Bio-one, 188271)

• 50 mL centrifuge tube, conical (Greiner Bio-one, 227261)

• 24-well plate TC-treated (Scientific Laboratory Supplies, 3526)

• DPBS, no calcium, no magnesium (ThermoFisher, 14190144)

• 16% paraformaldehyde (ThermoFisher, 11586711)

• Triton-X-100 (Sigma-Aldrich, T8787-50ML)

• Donkey serum (Sigma-Aldrich, D9663)

• DAPI (Hoechst) (Bio-Techne, 5748/10)

This protocol is split into 4 sections:

1: Cell fixation

2: Blocking & permeabilization

3: Primary antibody labelling

4: Secondary antibody labelling

1. Cell fixation

1.1. Prepare the following reagents, according to the instructions in Appendix 1:

1.1.1. 4% paraformaldehyde/PBS

1.1.2. 0.1% Triton-X-100/DPBS

1.1.3. Blocking Solution

1.2. Carefully remove spent culture medium, without disturbing the cells.

1.3. Add 500 μL of DPBS to each well.

1.4. Incubate for 5 minutes at room temperature.

1.5. Remove the DPBS without disturbing the cells.

1.6. Carefully add 250 μL of cold 4% paraformaldehyde/PBS to each well.

1.7. Incubate at 4°C for 15 minutes.

1.8. Remove the solution from each well without disturbing the cells.

1.9. Carefully add 500 μL of DPBS to each well.

1.10. Incubate cells for 5 minutes at room temperature.

1.11. Repeat steps 1.8 to 1.10 once more, leaving the cells in 500 μL of DPBS.

1.12. If the staining will not proceed immediately, wrap the plate with parafilm and store at 4°C overnight. Otherwise, continue to the next section.

2. Blocking and permeabilisation

2.1. Carefully remove the DPBS.

2.2. Gently add 500 μL of blocking solution down the side of each well.

2.3. Incubate at room temperature for 1 hour.

3. Primary antibody labelling

3.1. Prepare the primary antibody solutions described in Appendix 1.

3.2. Following the incubation described in step 2.3, aspirate blocking solution.

3.3. Carefully add 500 μL of the primary antibody mixture to the appropriate wells.

3.4. Add 500 μL of 0.1% TritonX-100/PBS to the negative control wells.

3.5. Seal plates with parafilm and incubate overnight at 4°C.

3.6. Remove the liquid from each well without disturbing the cells.

3.7. Carefully add 500 μL of DPBS to each well.

3.8. Incubate cells for 5 minutes at room temperature.

3.9. Repeat steps 3.6 to 3.8 a further 2 times, leaving the cells in 500 μL of DPBS before moving on to secondary antibody labelling

4. Secondary antibody labelling:

4.1. Meanwhile, prepare the secondary antibody solutions described in Appendix 1.

4.2. Aspirate the DPBS from the wells.

4.3. Carefully add 500 μL of secondary antibody (with DAPI) to the appropriate wells.

4.4. Incubate cells with the secondary antibody mixture for 1 hour at room temperature.

4.5. After incubation, remove the liquid from each well without disturbing the cells.

4.6. Carefully add 500 μL of DPBS to each well.

4.7. Incubate cells for 5 minutes at room temperature - wrap the plates in foil or place them in a cupboard to prevent fluorophore bleaching.

4.8. Repeat steps 4.5 to 4.7 a further 2 times, leaving the cells in 500 μL of DPBS.

4.9. Image each well using a fluorescent microscope with the fluorescent channel most appropriate for each antibody.

Preparation of 4% paraformaldehyde/PBS:

1. Add 30 mL of DPBS to a 50 mL centrifuge tube.

2. Add 10 mL of 16% paraformaldehyde.

3. Mix gently and store in the fridge until use.

Preparation of 0.1% Triton-X-100/DPBS:

1. Add 500 μL of Triton-X-100 stock solution to 500 mL of DPBS.

2. Mix thoroughly and store at room temperature until use.

3. Preparation of blocking solution:

4. Add 29.1 mL of 0.1% Triton-X-100/DPBS to a 50 mL centrifuge tube.

5. Add 900 μL of donkey serum.

6. Mix gently and store in the fridge until use.

Preparation of primary and secondary antibody solutions:

1. Prepare the base antibody solution by diluting 1 mL of blocking solution in 9 mL of DPBS.

2. Refer to the table below for antibody dilution recommendations.

3. Dilute each antibody in a relevant volume of the base antibody solution.

Table 1: Validated antibody information for the general characterisation of ioGABAergic Neurons.

If you have any questions or need assistance, please reach out to technical@bit.bio and we will do our best to support you.

Published October 2023, version 1