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Fixation methods can differentially affect ciliary protein immunolabeling

Overview of attention for article published in Cilia, March 2017
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  • Above-average Attention Score compared to outputs of the same age (63rd percentile)

Mentioned by

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4 tweeters

Citations

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17 Dimensions

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97 Mendeley
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Title
Fixation methods can differentially affect ciliary protein immunolabeling
Published in
Cilia, March 2017
DOI 10.1186/s13630-017-0045-9
Pubmed ID
Authors

Kiet Hua, Russell J. Ferland

Abstract

Primary cilia are immotile, microtubule-based organelles present on most cells. Defects in primary cilia presence/function result in a category of developmental diseases referred to as ciliopathies. As the cilia field progresses, there is a need to consider both the ciliary and extraciliary roles of cilia proteins. However, traditional fixation methods are not always suitable for examining the full range of localizations of cilia proteins. Here, we tested a variety of fixation methods with commonly used cilia markers to determine the most appropriate fixation method for different cilia proteins. Mouse inner medullary collecting duct and human retinal pigmented epithelial cells were grown to confluence, serum starved, and fixed with one of the following fixation agents: paraformaldehyde-sucrose, paraformaldehyde-PBS, methanol, cytoskeletal buffer followed by methanol, or three variations of cytoskeletal buffer-paraformaldehyde fixation. Each cell type and fixation method combination was probed with the following ciliary markers: acetylated α-tubulin, detyrosinated tubulin, polyglutamylated tubulin, β-tubulin, adenylyl cyclase 3 (AC3), ADP-ribosylation factor-like protein 13b (Arl13b), centrosome and spindle pole associated protein 1 (CSPP1), or intraflagellar transport protein 20 (IFT20). Intraflagellar transport protein 88 (IFT88) and GM130 (Golgi marker) were also used. We assessed actin (via phalloidin) and microtubule integrity, centrioles, cilia, and two extraciliary sites (mitotic figures and Golgi). For the cilia markers examined, paraformaldehyde fixation preserved cilia immunolabeling of cilia-membrane proteins (AC3 and Arl13b), but failed to reveal cilia immunostaining of axonemal proteins (CSPP1 and IFT20). Methanol revealed cilia labeling for some axonemal proteins, but not others, and this depended on cell type. Generally, any method that first included a wash in cytoskeletal buffer, before fixing, revealed more distinct cilia immunolabeling for axonemal proteins (CSPP1, IFT20, and IFT88), but resulted in the loss of cilia labeling for cilia-membrane proteins (AC3 and Arl13b). All three different post-translational modifications of tubulin antibodies positively immunolabeled cilia in all fixation methods tested. Ultimately, we found that fixing cells in a solution of paraformaldehyde prepared in cytoskeletal buffer allowed for the preservation of cilia immunolabeling for most cilia proteins tested and allowed visualization of two extraciliary sites (mitotic figures and Golgi). Some general patterns were observed to guide in the choice of a fixation agent. Cilia-membrane proteins generally benefit from quick fixation with no prior permeabilization, whereas axonemal proteins tend to benefit from permeabilization and use of cytoskeletal buffer.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 97 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Germany 1 1%
Unknown 96 99%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 29 30%
Student > Master 18 19%
Researcher 17 18%
Unspecified 9 9%
Professor > Associate Professor 9 9%
Other 15 15%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 39 40%
Agricultural and Biological Sciences 25 26%
Unspecified 12 12%
Neuroscience 10 10%
Medicine and Dentistry 6 6%
Other 5 5%

Attention Score in Context

This research output has an Altmetric Attention Score of 3. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 30 March 2017.
All research outputs
#2,478,008
of 9,269,265 outputs
Outputs from Cilia
#20
of 72 outputs
Outputs of similar age
#91,677
of 260,499 outputs
Outputs of similar age from Cilia
#1
of 1 outputs
Altmetric has tracked 9,269,265 research outputs across all sources so far. This one has received more attention than most of these and is in the 59th percentile.
So far Altmetric has tracked 72 research outputs from this source. They receive a mean Attention Score of 2.7. This one has gotten more attention than average, scoring higher than 72% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 260,499 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 63% of its contemporaries.
We're also able to compare this research output to 1 others from the same source and published within six weeks on either side of this one. This one has scored higher than all of them