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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.
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.
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....
1. Preparation of culture media, solutions, and dishes
The immunofluorescent staining of primary cilia is a relatively simple procedure that results in high-quality images. In these experiments, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope following the protocol described above. The primary cilium was detected using acetylated α-tubulin and γ-tubulin. The evaluation of primary cilia can be performed on various levels and any change in this regard can be linked to exposure to ionizing radiation, c.......
Several authors have described diverse methods for the detection of primary cilia, sometimes also describing various fixation methods that can affect their detection6,20,21,22. 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.......
This work was supported by the Ministry of Defence of the Czech Republic -Â Long-term organization development plan Medical Aspects of Weapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defence; the Ministry of Education, Youth and Sport, Czech Republic (Specific Research Project No: SV/ FVZ201703) and PROGRES Q40/06. Thanks also to Daniel Diaz for his kind assistance in English language revision.
....Name | Company | Catalog Number | Comments |
6-well plate | TPP | 92406 | Dimensions 128x86x22 mm |
Alexa Fluor488 | Jackson ImmunoResearch | 111-546-047 | AffiniPure F(ab')â‚‚ 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 |
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