Simple Detection of Primary Cilia by Immunofluorescence

Alzbeta Filipova1, Daniel Diaz Garcia2, Josef Dvorak3, Stanislav Filip4, Marcela Jelicova1, Zuzana Sinkorova1
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Filipova, A., Diaz Garcia, D., Dvorak, J., Filip, S., Jelicova, M., Sinkorova, Z. Simple Detection of Primary Cilia by Immunofluorescence. J. Vis. Exp. (159), e61155, doi:10.3791/61155 (2020).

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 ionizing radiation3,4,5,6,7. Usually, cells have only one primary cilium, which is involved in morphogenesis and cell signalling pathways important for cell proliferation and differentiation8,9.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases, and resorbed before entering mitosis in a process associated with tubulin deacetylation mediated by HDAC6 (histone deacetylase 6)10. The exact moment of primary cilia resorption depends upon cell type and the expression of genes directly involved in this process, such as Aurora A, Plk1, TcTex-111,12,13. Depending on the cell type, the primary cilia express different types of receptors, ion channels, and active signalling pathways. These include the most important signalling receptors affecting proliferation and survival, EGFR, PDGFR, and FGFR. Also included are some of the signalling pathways that may affect the function of one or more organs, including Hedgehog, Notch, and Wnt. Thanks to these receptors and signalling pathways, the primary cilia also perform a chemosensory function. This function allows primary cilia to detect specific ligands for Notch, hormones, and biologically active substances such as serotonin or somatostatin. Other specific functions exhibited by primary cilia of different lengths include reaction to changes in temperature, gravity, and osmolality14.

Primary cilia can be visualized through various methods, such as live visualization, transmission electron microscopy, 3D imaging, or by software for the automatic detection of primary cilia5,15,16,17. However, these methods are highly specialized and ongoing research needs basic, fast, and easy methods for staining primary cilia in every stage of research. Described is an easy and useful method for the detection of primary cilia in cultured cells.

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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 experiment. Stir and heat the solution at 55 °C for 30 min in the hood. Cool down at RT. Add 1 M sodium hydroxide until the solution becomes clear (pH = 7.2–7.4). Store at 4 °C for up to 1 week.
    Note: PFA is toxic; always wear adequate personal protective equipment and prepare in the chemical hood.
  3. Prepare 500 mL of culture media, DMEM (Dulbecco´s Modified Eagle´s medium) containing 10% FBS, 1% penicillin/streptomycin, and 2% glutamine.
  4. Prepare 13 mL of 1% gelatin solution in sterile dH2O (130 mg of gelatin in 13 mL of dH2O). Use 2 mL of 1% gelatin for each well in a 6 well plate. Keep sterile.
  5. Clean the laminar flow hood using 70% ethanol. Place the required material inside the laminar flow hood before starting the experiment.

2. Cell culture for immunocytochemistry staining

  1. Thaw the cells (in this study C2C12, MEF, NHLF, and skin fibroblasts) using standard techniques and plate them in a T75 flask supplemented with ~10–12 mL of the prepared media. Incubate at 37 °C/5% CO2/90% relative humidity (RH) until the cells reach 70% confluence.
  2. Remove the cells from the incubator and place them in the laminar flow hood. Remove the culture media and rinse the cells briefly 2x with 1x PBS. Add ~2 mL of 0.25% trypsin-EDTA into the T75 flask and incubate at 37 °C for ~5 min. Check periodically on the inverted microscope to monitor cell detachment.
    NOTE: The incubation time depends on the cell line and therefore must be determined empirically.
  3. Gently resuspend the cells in 10 mL of culture media, pipetting carefully to create a single cell suspension. Rinse the flask again if necessary.
  4. Place the cell suspension in a 50 mL conical tube and centrifuge for 5 min at ~200 x g. Decant the supernatant, add 10 mL of culture media, and gently resuspend the pellet. Take 20 µL of the cell suspension and mix in a 1:1 ratio with trypan blue and count in a cytometer following the standard method.
  5. Place one coverslip inside each well of a 6 well plate using tweezers. Coat the coverslips with gelatin by pouring ~2 mL into the wells. This will help the cells attach to the coverslips. Remove the gelatin solution and let air-dry for a few minutes. The coverslips are now ready for cultivation of the cells. Start the cultivation immediately.
  6. Seed 100,000 fibroblasts into each well and add 2 mL of culture media. Incubate the cells for 24 h at 37 °C/5% CO2/90% RH. At this point, the cells can be treated according to the needs of the user. Treatments to induce ciliation have been previously described4,5.
    NOTE: The initial seeding number depends on the cells’ doubling time and should be determined accordingly.

3. Immunofluorescent staining of primary cilia in vitro

  1. Warm the 4% paraformaldehyde to RT. Prepare Pasteur pipettes, 1x PBS (RT), waste container, 15 mL conical tubes, micropipettes (0.5–10 µL, 20–200 µL, and 100–1,000 µL) and tips. Take the cells from the incubator and place them in the bench.
    NOTE: The staining procedure does not need to be performed in sterile conditions. All solutions must be at RT.
  2. Remove media from each well. Leave the coverslip inside the well. Very gently wash the cells 3x with 2 mL of 1x PBS. Using a Pasteur pipette, add 2 mL of 4% PFA into each well to fix the cells. Incubate for 10 min at RT. Remove the PFA and wash 3x with 1x PBS.
    NOTE: Always use a sufficient volume to cover the entire coverslip during the incubation periods. Never let the cells dry. Never pour any of the solutions directly onto the coverslip.
  3. Prepare 0.5% Triton X-100 in 13 mL of 1x PBS 10 min before use. Add 2 mL into each well. Incubate for 15 min. Wash gently 4x with 1x PBS.
    NOTE: Triton X-100 is insoluble in PBS at RT. Heat the 0.5% Triton X-100 solution to 37 °C in a water bath to dissolve it.
  4. Thaw goat serum 5 min before use. Dilute the goat serum in 1x PBS in a 1:20 ratio as a blocking solution. Add 150 µL to each coverslip and incubate for 20 min at RT.
    NOTE: Prolong the blocking period up to 60 min if necessary. Do not wash the cells after blocking with goat serum.
  5. Thaw the primary antibodies (i.e., anti-acetylated alpha tubulin and anti-gamma tubulin) 5 min before use. Dilute the antibodies separately in 1x PBS as follows: mouse anti-acetylated alpha tubulin in a 1:800 ratio and rabbit anti-gamma tubulin in a 1:300 ratio. Remove the blocking solution. Do not wash. Add 150 µL of both antibody dilutions to the coverslips and incubate for 60 min at RT.
    NOTE: If incubating overnight use 500–1,000 µL of the primary antibody solutions, seal the 6 well plate with paraffin film, and store at 4 °C. Alternatively, use 150 µL of antibody and incubate in a humidity chamber.
  6. Remove the primary antibodies. Wash the coverslips very gently 3x with 2 mL of 1x PBS. Prepare the secondary antibodies in 1x PBS by separately diluting Cy3 sheep anti-mouse and Alexa Fluor488 goat anti-rabbit in a 1:300 ratio. Add 150 µL of both secondary antibody dilutions to the coverslips. Incubate for 45 min at RT in the dark.
    NOTE: Incubate in the dark to avoid photobleaching. Other combinations of secondary antibodies can be used as needed.
  7. Prepare a DAPI (4', 6-diamidino-2-phenylindole) solution according to the manufacturer's instructions. Store the excess aliquots at -20 °C. Dilute 10 µL from a stock aliquot (1:5,000) in 50 mL of 1x PBS. Add 2 mL of this dilution to the coverslips. Incubate for 5 min at RT in the dark.
    NOTE: It is important to incubate the cells in the dark to avoid photobleaching. The DAPI dilution can be stored at 4 °C for up to 1 month.
  8. Prepare 2 needles, slides, tweezers, and mounting media. Label the slides.
  9. Remove the DAPI solution from the wells. Wash 3x with 1x PBS. Put one drop of mounting media on each slide. Use the needle to gently lift the coverslip from the well’s bottom. Flip the coverslip using the tweezers and gently place it over the drop of mounting media. Carefully remove any bubbles.
  10. Protect the slides from light and store them overnight at 4 °C.
  11. Use a fluorescent or confocal microscope with high magnification to visualize the primary cilia.
    NOTE: The slides can be stored in the dark at 4 °C for up to 2 months.

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Representative Results

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, cell metabolism (e.g., starvation), or chemical treatment (e.g., cytostatics)5,18.

The effect of ionizing radiation on primary cilia has been studied in various cell lines (e.g., the myoblast cell line C2C12), which were irradiated (2, 6, 10, and 20 Gy) and the changes in primary cilia incidence analyzed. According to Filipova at al.4, low irradiation doses do not modify the occurrence of a single primary cilia in C2C12 cells. However, higher doses of ionizing radiation (i.e., 20 Gy) induced the appearance of multiple primary cilia (Figure 1A,B,C). Similarly, when NHLF cells were irradiated at 2 Gy the primary cilia were detected by immunofluorescence (Figure 2).

Metabolic stress is also known to increase the frequency of primary cilia19. In this case, MEF fibroblasts were starved and analyzed for changes in primary cilia incidence (Figure 3).

Immunofluorescence staining revealed that fibroblast cells carried primary cilia after treatment with doxorubin and taxol. Those fibroblasts treated with 120 nM doxorubicin expressed a single primary cilium (Figure 4); higher doses induced the appearance of multiple primary cilia (Figure 5). Treatment with 1.25 nM taxol also resulted in the presence of a single primary cilium (Figure 6). In contrast to the treatment with doxorubicin, multiple cilia were not detected after treatment with higher doses of taxol5.

Figure 1
Figure 1: Occurrence of primary cilia in irradiated C2C12 cells. Representative photographs of primary cilia in C2C12 cells. Primary cilia detection was performed by immunofluorescence. The axoneme (arrow) of the primary cilia were assessed with acetylated α-tubulin antibody (red) and the basal body by γ-tubulin antibody (arrow, green). Nuclei were stained with DAPI (blue). (A) and (B) multiple cilia were observed 72 h after irradiation with 20 Gy. (C) Single primary cilia after 72 h irradiation with 20 Gy4. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Detection of primary cilia in irradiated NHLF cells. Representative photographs of primary cilia in NHLF cells. Primary cilia (arrow) detection was performed by immunofluorescence. The axonemes of the primary cilia were stained with acetylated α-tubulin antibody (red) and the basal bodies with γ-tubulin antibody (green). Nuclei were stained with DAPI (blue). Single primary cilia 24 hours after irradiation at 2 Gy. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Incidence of primary cilia in the MEF cells after metabolic stress induced by serum starvation. Representative photographs of primary cilia 24 h after serum starvation (0.1% FBS) in MEF cells. Primary cilia (arrow) detection was performed by immunofluorescence. Axonemes were labeled with acetylated α-tubulin antibody (red). Basal bodies were stained with γ-tubulin antibody (green). Nuclei were stained with DAPI (blue). Please click here to view a larger version of this figure.

Figure 4
Figure 4: Representative photographs of primary cilia in skin fibroblasts. Primary cilia (arrow) detection was performed by immunofluorescence. Primary cilia were stained with acetylated α-tubulin antibody (red), while the basal bodies were stained with γ-tubulin antibody (green). Nuclei were stained with DAPI (blue). Primary cilia were detected 72 h after treatment with 120 nM doxorubicin5. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Representative photographs of multiple cilia in skin fibroblasts. Primary cilia (arrow) detection was performed by immunofluorescence. The axonemes were labeled by acetylated α-tubulin antibody (red) and the basal bodies were stained with γ-tubulin antibody (green). Nuclei were stained with DAPI (blue). Multiple cilia were detected 72 h after treatment with 120 nM doxorubicin5. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Representative photographs of skin fibroblasts treated with taxol. Primary cilia (arrow) were detected by immunofluorescence. Primary cilia were stained with acetylated α-tubulin antibody (red) and with γ-tubulin antibody (green). Axoneme nuclei were stained with DAPI (blue). Primary cilia were detected 72 h after treatment with 1.25 nM taxol5. Please click here to view a larger version of this figure.

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Discussion

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, especially in early stages of research or for a quick and easy method to test the presence of primary cilia in a chosen cell line. Therefore, this protocol is described in as much detail as possible for the detection of primary cilia in vitro after different kinds of treatment.

The present protocol was modified for use on a daily basis20,23,24. For example, 10% formalin was replaced by 4% PFA, whose fresh preparation is recommended due to its short storage life. PFA is a good choice for preserving cell morphology and is especially suited to the visualization of membrane-bound proteins. Organic solvents, such as methanol, have a dehydrating effect on the cell and remove small, soluble molecules and lipids during the fixation process, thus making it unsuitable for use in certain scenarios25. Permeabilization is achieved with 0.5% Triton X-100 in 1x PBS for 15 min. Goat serum in a 1:20 dilution in 1x PBS for 20 min is used as a blocking agent. Both primary antibodies, mouse anti-acetylated tubulin and rabbit anti-γ-tubulin, can be incubated concurrently for 60 min using a 1:800 and 1:300 dilution in 1x PBS, respectively20,21,23,24. In addition, the secondary antibodies, anti-mouse IgG (whole molecule) F(ab′)2 fragment–Cy3 antibody produced in sheep and Alexa Fluor488 AffiniPure F(ab')₂ fragment goat anti-rabbit IgG, were diluted 1:300 in 1x PBS. They were incubated concurrently for 45 min.

It may be necessary to take extra standardization steps should the primary antibodies be incubated overnight. During the development of the protocol it was found that an overnight incubation needs a volume of at least 500–1,000 µL of primary antibodies solution, the 6 well plate must be sealed with parafilm, and storage must be at 4 °C to prevent evaporation.

The most critical steps for the successful staining of primary cilia are: 1) choice of cell line and optimal cell culture practice; 2) use of gelatin coated coverslips; 3) consistent use of fresh 4% paraformaldehyde; 4) incubation of the secondary antibody and DAPI in the dark; 5) performing a gentle flip and placement of the coverslip on top of the mounting media in the slide.

There are no foreseen potential limitations in the future applications of the protocol. Moreover, primary cilia research is becoming more relevant in a variety of fields, and easy, fast, and reliable cilia detection methods are essential. Further, this protocol will facilitate the future study of primary cilia in cell types in which primary cilia have been heretofore undetected.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was supported by the Ministry of Defence of the Czech Republic - 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.

Materials

Name Company Catalog Number Comments
6-well plate TPP 92406 Dimensions 128x86x22 mm
Alexa Fluor488 Jackson ImmunoResearch 111-546-047 AffiniPure F(ab')? 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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References

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