Multifluorescence labeling techniques and confocal laser scanning microscopy on lung tissue

Our plain-English summary of Multifluorescence labeling techniques and confocal laser scanning microscopy on lung tissue, by M. Stern, D.J. Taatjes, B.T. Mossman (Methods in Molecular Biology, 2006).

The study

• Authors: M. Stern, D.J. Taatjes, B.T. Mossman
• Journal: Methods in Molecular Biology
• Published: 2006;319:67-76
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Lung tissue consists of more than 40 individual cell types that might interact to produce adverse pathologies. After injury, a number of signaling proteins expressed in various epithelial and other cell types have been linked to the advent of apoptosis, compensatory proliferation, and adaptation to stress. We describe here the use of immunochemistry and multifluorescence approaches using confocal laser scanning microscopy to define the signaling pathways (protein kinases C and mitogen-activated protein kinases) activated by asbestos fibers after inhalation. Using these approaches, we are able to localize signaling events in distinct cell types of the lung and determine their status in the cell cycle (resting or nonresting). Moreover, we are able to determine whether various signaling proteins colocalize in cells and the sites affected by asbestos fibers.

In plain English

This was a laboratory methods chapter, not a study reporting new results. The authors gave step-by-step procedures for staining frozen mouse lung tissue with several fluorescent antibody labels at once and viewing it with confocal laser scanning microscopy. The procedures aimed to document activation of two signaling proteins linked to cell growth and cell death, PKCδ and ERK1/2, in the lung after asbestos was inhaled. A staining procedure for a third protein, JNK, was also given. The authors also described how to identify which cell types carried these proteins, using markers for epithelial cells, alveolar type II cells and macrophages. The Ki-67 marker showed whether cells were resting or nonresting (proliferating). One figure showed triple labeling in sham animals and in animals exposed to crocidolite asbestos for 4 days, plus a control slide that showed no signal. The caption did not describe differences between the groups, and no counts or measurements were given. Statements about asbestos effects on these proteins came from earlier studies the chapter cited. The authors stated that these approaches let them localize signaling events in distinct lung cell types.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods chapter (staining and microscopy protocol); no new study results reported
• Who was studied: Lung tissue from laboratory mice; number of animals, location and dates not given
• Exposure: Inhaled asbestos in general. The example figure used animals exposed to crocidolite asbestos for 4 days, compared with sham animals. No dose was given.
• Main results:
  • No measured results were reported. Figure 1 illustrated triple labeling for PKCδ, p-ERK and Ki-67 in sham animals and in animals exposed to crocidolite asbestos for 4 days. White areas marked where all three labels overlapped. A negative control without primary antibody showed no fluorescence signal. The caption did not describe differences between exposed and sham animals.
• The authors' conclusion: The authors stated that these methods allowed them to localize signaling events in distinct cell types of the lung. They also stated that the methods could show whether cells were resting or nonresting, whether signaling proteins colocalized, and which sites were affected by asbestos fibers.
• Limitations the authors state:
  • Technical caution: when two or three antibodies are combined, different antigens may need different fixation conditions
  • Technical caution: lung tissue gives off its own background glow (autofluorescence). The authors advised using probes that fluoresce at higher wavelengths to reduce it.

Topics

Medical terms in this study, explained in our Lexicon: Apoptosis.

Related studies

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• Environmental scanning electron microscopy technique to identify asbestos phases inside ferruginous bodies — A. Croce, M. Musa, et al. (2013)
• Final results from a 90-day quantitative inhalation toxicology study evaluating the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos: Histopathological examination, confocal microscopy and collagen quantification of the lung and pleural cavity — D.M. Bernstein, B. Toth, et al. (2021)
• Evaluation of the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos in a 90-day quantitative inhalation toxicology study - Interim results Part 2: Histopathological examination, Confocal microscopy and collagen quantification of the lung and pleural cavity — D.M. Bernstein, B. Toth, et al. (2020)
• Confocal Laser Endomicroscopy as a Guidance Tool for Pleural Biopsies in Malignant Pleural Mesothelioma — L. Wijmans, P. Baas, et al. (2019)

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