Using immunocytochemistry (ICC) researchers can detect relevant epithelial markers and assess for markers of differentiation. Besides other end-point characterization studies, ICC staining also enables researchers to evaluate different experimental conditions. For instance, it can be used to assess culture marker expression in the presence or absence of a specific drug or pathogen.

Below, we describe a protocol for fixation and immunostaining of pluripotent stem cells (PSC)-derived kidney organoids using the STEMdiff™ Kidney Organoid Kit (Catalog# 05160). For immunostaining other epithelial cell types, please refer to our protocol Performing Immunocytochemical Staining of Epithelial Organoids.

Materials

• D-PBS (Without Ca++ and Mg++; Catalog #37350)
• Paraformaldehyde (e.g. Affymetrix 199431LT)
• Triton™ X-100 (e.g. Millipore Sigma X100)
• Donkey Serum (eg. Millipore Sigma D9663)
• DAPI (Catalog #75004)

Preparation

Prepare the following reagents before proceeding to fixation and immunostaining:

1. Prepare a 4% solution of paraformaldehyde in D-PBS. Mix thoroughly and store at 2 - 8°C.
2. Prepare the PBS-T (permeabilization buffer) by diluting Triton™ X-100 in D-PBS to a final concentration of 0.2%. Mix thoroughly.
   Note: Prepare fresh for each experiment.
3. Prepare blocking buffer by diluting Donkey Serum in PBS-T to a final concentration of 10%. Mix thoroughly. Store on ice or at 2 - 8°C while in use.
   Note: Prepare fresh for each experiment.
4. Prepare the DAPI Solution by adding DAPI to Blocking Buffer to a final concentration of 1 µg/mL. Mix thoroughly.

Protocol

1. Aspirate medium from the well containing kidney organoids. Carefully add 200 µL D-PBS to the well. Remove D-PBS.
2. Add 85 µL 4% Paraformaldehyde Solution to the well (fixation). Incubate at room temperature (15 - 25°C) for 15 minutes. Remove the solution.
3. Wash the well 3X with 200 µL D-PBS. Remove D-PBS.
   Note: For later immunostaining, keep 200 µL D-PBS in the well, wrap plate with Parafilm®, and store at 2 - 8°C. Remove remaining D-PBS before proceeding to Step 4.
4. Add 200 µL PBS-T to the well. Incubate at room temperature (15 - 25°C) for 15 minutes. Remove PBS-T.
5. Add 200 µL Blocking Buffer to the well. Incubate at room temperature (15 - 25°C) for at least 30 minutes.
6. While incubating Blocking Buffer in the well, prepare primary antibody mix by diluting each primary antibody in Blocking Buffer. Refer to Table 1 for recommended dilutions and antibodies.
7. Remove Blocking Buffer from the well and add 80 µL primary antibody mix. Incubate at 2 - 8°C overnight with low shaking.
8. Prepare secondary antibody mix by diluting each secondary antibody 1 in 1000 in Blocking Buffer + DAPI (1 µg/mL final concentration). Refer to Table 1 for recommended antibodies.
9. Add 200 µL D-PBS to the well and incubate at room temperature (15 - 25°C) for 5 minutes. Remove D-PBS. Repeat this wash step 5X for a total of 6 washes.
10. Add 80 µL secondary antibody mix to the well. Incubate in the dark at room temperature (15 - 25°C) overnight with low shaking. Remove secondary antibody mix.
11. Add 200 µL D-PBS to the well and incubate at room temperature (15 - 25°C) for 5 minutes. Remove D-PBS. Repeat this wash step 5X for a total of 6 washes.
12. Add 200 µL D-PBS to stained cells; they are now ready for immunofluorescent imaging.
    Note: If not used immediately for imaging, wrap plate with Parafilm® and store in the dark at 2 - 8°C.