Simple Detection of Primary Cilia by Immunofluorescence

Summary

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Abstract

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Introduction

Primary cilia are sensory, solitary, membrane-bound, nonmotile structures associated with the cell’s mother centriole. Primary cilia are found on most vertebrate cells with the exception of red blood cells, adipocytes¹, and hepatocytes². Primary cilia are formed as an elongated axoneme composed by microtubules, whose main component is α-tubulin. The axoneme grows from the basal body, which is structured from γ-tubulin. The length of the primary cilia varies between 2–10 µm; however, its dimensions can change during glycylation, starvation, hypoxia, cytotoxic stress, or after exposure to ionizin....

Protocol

1. Preparation of culture media, solutions, and dishes

1. Autoclave the coverslips (22 x 22 mm). Prepare 6 well plates. Thaw fetal bovine serum (FBS) and antibiotic penicillin/streptomycin and warm the culture medium to room temperature (RT). Use trypsin-EDTA (0.25%) and 1x PBS (phosphate buffered saline with calcium and magnesium) to passage the cells.
2. Prepare fresh 4% paraformaldehyde (PFA) in dH₂O (800 mg of PFA in 20 mL of dH₂O). The PFA must be freshly prepared for each exper.......

Discussion

Several authors have described diverse methods for the detection of primary cilia, sometimes also describing various fixation methods that can affect their detection^6,20,21,22. Regardless, it is difficult to find a complete and straightforward protocol for detection. The ready availability of such a method would undoubtedly be of great assistance to the study of primary cilia investigation, esp.......

Materials

Name	Company	Catalog Number	Comments
6-well plate	TPP	92406	Dimensions 128x86x22 mm
Alexa Fluor488	Jackson ImmunoResearch	111-546-047	AffiniPure F(ab')₂ Fragment Goat Anti-Rabbit IgG
Anti-Tubulin γ	Sigma-Aldrich	T5192	Polyclonal Rabbit anti-Mouse IgG2a
C2C12	ATCC	CRL-1772	Myoblast (mouse)
Cy3	Sigma-Aldrich	C2181	Anti-Mouse IgG (whole molecule) F(ab′)2 fragment–Cy3 antibody produced in sheep
Dapi (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma-Aldrich	D9542
Dulbecco´s Modified Eagle´s medium	Thermo Scientific	11960044	High glucose, No glutamine, Gibco
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8662	With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture
Fetal Bovine Serum	Thermo Scientific	16000044	Sterile-Filtered, Gibco
L-Glutamine	Sigma-Aldrich	G7513
MEF	ATCC	SCRC-1039	Mouse embryonic fibroblast
Monoclonal Anti-Acetylated Tubulin	Sigma-Aldrich	T7451	Monoclonal Anti-Acetylated Tubulin antibody produced in mouse
NHLF	Lonza	CC-2512	Primary lung fibroblasts (human)
Normal Goat Serum	Jackson ImmunoResearch	005-000-121
Paraformaldehyde	Sigma-Aldrich	158127-500G	Powder
Penicillin-Streptomycin	Sigma-Aldrich	P0781	10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered
ProLong Diamond Antifade Mountant	Thermo Scientific	P36961
Skin fibroblasts	Kindly gifted from Charles University, Faculty of Medicine in Hradec Králové.
Square Cover Slips	Thermo Scientific	22X22-1.5	Borosilicate glass, 22x22mm, Square
Triton X-100	Sigma-Aldrich	11332481001
Trypsin-EDTA (0.25%)	Thermo Scientific	25200072	Sterile-Filtered, Gibco