Immunocytochemical staining protocol for human iPSC-derived glutamatergic neurons

Immunocytochemical staining protocol for human iPSC-derived glutamatergic neurons

Protocol overview

Pictorial workflow of the steps involved in the ioGlutamatergic Neuron ICC analysis.

Introduction

Immunocytochemistry (ICC) staining is a powerful tool that enables specific proteins or other molecules to be quantitatively visualised in a cellular context. As such, ICC can provide insights into the distribution, localisation, and abundance of specific proteins or other molecules. 

There are many valuable applications of ICC in research and drug discovery. For example, ICC staining can be used to investigate the absence and potential therapeutic restoration of the DMD protein in cellular models of Duchenne muscular dystrophy- as was achieved with ioSkeletal Myocytes DMD Exon 44 Deletion™. Additionally, high content analysis of ICC experimental data can be used to develop phenotypic screening assays. In this way, ICC has been used to quantify specific protein levels across multiple cultures—a process recently used to measure the hyperphosphorylation of Tau in a model of frontotemporal dementia in ioGlutamatergic Neurons™.

ICC is a versatile and widely used technique that provides researchers with valuable information about the structure, function, and behaviour of cells. While ICC staining of iPSC-derived cells is a relatively straightforward process, it does require careful consideration of antibody pairings, delicate treatment of cells, and optimisation to reduce non-specific signals. Here, we provide an optimised protocol for the ICC staining of ioGlutamatergic Neurons.

The following protocol recommends general guidelines. We encourage users  to optimise the critical steps according to their experimental conditions.

This protocol is specifically designed for 24-well plates. If using another plate format, please refer to the suppliers information for the recommended media volumes.

 

Materials and equipment

Protocol

This protocol is split into 4 sections:

1: Cell fixation

2: Blocking & permeabilization

3: Primary antibody labelling

4: Secondary antibody labelling


Throughout this protocol, use a micropipette to remove liquids from each well, making sure not to disturb the cell layer.

Do not allow the cell layer to dry out; leave behind approximately 50 μL in the well after removing media.

1. Cell fixation

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

1.1.1. 4% paraformaldehyde/PBS

Paraformaldehyde is toxic by ingestion, inhalation, and absorption. Refer to the suppliers MSDS for paraformaldehyde and adhere to all recommendations before proceeding.

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.

Neuronal cells are sensitive to mechanical stress. Perform all media additions slowly and on the side of the well.

Always use micropipettes, not serological pipettes, to prevent cell detachment.

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.

Protect the plate from light to prevent fluorophore bleaching; cover plates with tin foil.

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.

Appendix 1

Preparation of 4% paraformaldehyde/PBS:

Any handling of paraformaldehyde should be performed in an appropriate safety cabinet. Refer to the paraformaldehyde SDS for specific handling instructions.

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 ioGlutamatergic Neurons.

Antibody

Supplier

Cat. Number

Storage temp.

Species

Dilution

Anti-Vesicular Glutamate Transporter 2 Antibody (VGLUT2)

Millipore

MAB5504

2°C to 8°C

Mouse

1/500

Anti-MAP2 antibody

Abcam

ab5392

2°C to 8°C

Chicken

1/2000

Purified anti-Tubulin β 3 (TUBB3) Antibody

Biolegend

801202H

2°C to 8°C

Mouse

1/1000

Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488

Thermo Fisher

A-21202

2°C to 8°C

Donkey

1/1000

Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor™ 647

Thermo Fisher

A-21449

2°C to 8°C

Goat

1/1000

DAPI

Bio-Techne

5748/10

-20°C

-

1/500

 

Application support

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

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