Celsr1 Immunostaining

Immunostaining – Celsr1

Sara N. Stahley, Danelle Devenport

Sara N Stahley https://orcid.org/0000-0002-2316-8354

Danelle Devenport https://orcid.org/0000-0002-5464-259X

*For correspondence:  danelle@princeton.edu

An abbreviated version of this protocol was published in eLife in February 2021. Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes, DOI: https://doi.org/10.7554/eLife.62097.

Detailed protocol

Abstract     

Celsr1 is an atypical cadherin that mediates the asymmetric organization of Frizzled6 and Vangl2 at cell-cell junctions enriched along the anterior-posterior body axis, which is essential for establishing planar cell polarity. Here, we describe a procedure utilizing immunofluorescence to detect both exogenous Celsr1 in cultured keratinocytes and endogenous Celsr1 in mouse epidermis.

Keywords: keratinocytes, epidermis, cadherin, planar polarity

Materials and Reagents

1. Celsr1-mNeonGreen mouse CD1 keratinocytes (Heck and Devenport, 2017)
2. E15.5 dorsal mouse epidermis (or desired embryonic stage)
3. E-medium supplemented with 15% FBS (chelated) and 0.05 µm Ca²⁺ (Nowak and Fuchs 2009)
4. 12 Well Tissue Culture Plate (CELLTREAT, Cat #229112)
5. 18 mm #1.5 glass coverslips (Warner Instruments, Cat #64-0714)
6. Microscope slides (Fisherbrand™ Premium Superfrost™, Cat #12-544-7)
7. Fibronectin (Corning, Cat #CB-40008B)
8. PBS+ (DPBS, calcium, magnesium; Gibco™, Cat #14040216)
9. PBS (DPBS, no calcium, no magnesium; Gibco™, Cat #14190235)
10. 16% Paraformaldehyde Aqueous Solution, EM Grade (Electron Microscopy Sciences, Cat #50-980-489)
11. Triton X-100 (Sigma, Cat #X100-100ML)
12. Antibodies
    1. Primary: Guinea pig anti-Celsr1 (Devenport and Fuchs, 2008) (1:1000)
    2. Secondary: Donkey anti-guinea pig Alexa Fluor® 488 (Jackson ImmunoResearch, Cat #706-545-148) (1:2000) (or similar depending on experimental needs)
13. ProLong™ Gold Antifade Reagent (Invitrogen, Cat# P36930)

Equipment

• CO₂ incubator
• Fluorescence microscope
• Image acquisition software
• ImageJ software for additional image processing (as desired)

Procedure

Cultured keratinocytes

1. Prepare fibronectin-coated coverslips:
   1. Transfer sterile #1.5 glass coverslips to tissue culture plate wells.
   2. Add 800 µl of fibronectin (1:100 in PBS) and let sit for 45 mins.
   3. Aspirate fibronectin and wash coverslips twice with PBS.
2. Seed Celsr1-mNeonGreen cells onto coated coverslips and culture in E-medium at 5% CO₂ 37°C until desired confluence (~70-80%).
3. Calcium switch: aspirate medium and replace with E-medium containing 1.5 mM calcium to induce cell junction formation for 8-24 hr. 
4. Wash cells three times with PBS+.
5. Fix in 4% PFA (paraformaldehyde in PBS) for 10 min at room temperature (RT). 
6. Wash cells for 5 min three times with PBS.
7. Permeabilize in 0.1% PBT (Triton X-100 in PBS).
8. Transfer coverslips to parafilm, add 55 µl primary antibody (in PBT) for 1 hr at RT.
9. Wash cells for 5 min three times with 0.1% PBT.
10. Add 100 µl secondary (in PBT) (+ Hoechst if desired) for 30 min. *Keep in dark.
11. Wash cells for 5 min three times with 0.1% PBT.
12. Wash cells once with PBS for 5 min.
13. Mount coverslips, cell side down, on glass slide with 15-20 µl ProLong Gold (or other suitable mounting media) and allow to cure completely at RT.
14. Image on a suitable microscope with fluorescence imaging capabilities.

Whole mount embryonic mouse skin

1. Collect E15.5 (or desired embryonic stage) embryos in PBS+ to maintain calcium-dependent epidermal cell-cell adhesions.
2. Fix embryos in PFA+ (4% paraformaldehyde in PBS+) for 1 hr at RT (rocking). 
3. Dissect whole dorsal skin from fixed embryo.
4. Block/permeabilize skins in 1.5 mL tube (4-5 skins max per tube) in PBT2 (0.2% Triton X-100 in PBS) containing 2.5% normal donkey serum, 1% BSA and 1% fish gelatin for 2 hr at RT or overnight at 4°C (rocking).
5. Incubate skins in primary antibody (in block) overnight at 4°C (rocking).
6. Wash skins for 30 min three times in PBT2 at RT (rocking). 
7. Incubate skins with secondary antibody (+ Hoechst if desired) for 2-3 hr at RT or overnight at 4°C (rocking). 
8. Wash skins for 5 min three times with PBT2.
9. Wash skins for 5 min with PBS.
10. Mount skins, epidermis toward coverslip, in ~40 µL ProLong Gold (or other suitable mounting media) and allow to cure completely at RT.
11. Image on a suitable microscope with fluorescence imaging capabilities.