Figure 1.
Figure 1. The MPO::GFP construct drives fluorescent protein expression in neutrophils, which participate in a spontaneously resolving inflammatory response to injury. Zebrafish larvae were anesthetized by the addition of 4.2% tricaine to the medium in which they were maintained (E3). GFP was visualized by excitation at 492 nm. All photomicrographs were taken under air immersion using either 10×/0.3 NA or 20× ELWD/0.45 NA Plan Fluor objectives. No nonlinear normalization was performed. Confocal images (C, D) were taken using a 60×/1.40 NA oil immersion Plan Apo objective. (A-B) Photomicrographs of larvae at 56 and 96 hours show expression of GFP over the first days of zebrafish myelopoiesis. (C) Summed Z stacks of consecutive images through the intermediate cell mass (ICM) of a day 3 transgenic zebrafish stained by in situ hybridization for MPO and by immunohistochemistry for GFP. Images taken through the Alexa-488 and TRITC channels are shown, followed by a merged image. In all GFP-positive cells (green), some MPO in situ signal (red) can be seen. Examination of individual sections confirms colocalization of these markers. (D) Summed Z stacks of consecutive images through the ICM of a day 3 transgenic zebrafish stained by in situ hybridization for L-plastin and by immunohistochemistry for GFP. Images taken through the Alexa-488 and TRITC channels are shown, followed by a merged image. GFP-positive cells (green) do not coexpress L-plastin (red). Examination of individual sections confirms absence of colocalization of these markers. (E) Tailfins of anesthetized wild-type AB zebrafish were transected at 4 days after fertilization. At the time points indicated, fish were anesthetized, then fixed and stained as described in “Materials and methods.” The number of neutrophils participating in the inflammatory response (excluding cells in the posterior blood island) was assessed. Data shown are mean ± SEM, n = 3, with 5 replicates per experiment. (F) Tailfins of anesthetized transgenic zebrafish were transected at 4 days after fertilization. At the time points indicated, individual fish were anesthetized and imaged as described. The number of fluorescent neutrophils participating in the inflammatory response was assessed. Data shown are mean ± SEM (n = 5). (G) Images taken for the counts shown in panel E were processed (IPlab) to quantify the total area of fluorescence. For each image, a series of fluorescence images in Z were projected into a single image using the maximum intensity at each point. These images were normalized, and a region of interest was defined corresponding to the area of inflammation quantitated. Segmentation of this region was performed in IPlab, and the area of fluorescence was exported to Excel. The procedure was automated by use of an IPlab script. (H) Fluorescence counts of individual larvae at 6 and 24 hours after injury (hpi) are shown. The individual pattern of resolution is evident for each fish. (I) Images showing a single transgenic zebrafish larva at the time points indicated (hpi). The accumulation of fluorescent cells and their subsequent removal can be clearly seen.

The MPO::GFP construct drives fluorescent protein expression in neutrophils, which participate in a spontaneously resolving inflammatory response to injury. Zebrafish larvae were anesthetized by the addition of 4.2% tricaine to the medium in which they were maintained (E3). GFP was visualized by excitation at 492 nm. All photomicrographs were taken under air immersion using either 10×/0.3 NA or 20× ELWD/0.45 NA Plan Fluor objectives. No nonlinear normalization was performed. Confocal images (C, D) were taken using a 60×/1.40 NA oil immersion Plan Apo objective. (A-B) Photomicrographs of larvae at 56 and 96 hours show expression of GFP over the first days of zebrafish myelopoiesis. (C) Summed Z stacks of consecutive images through the intermediate cell mass (ICM) of a day 3 transgenic zebrafish stained by in situ hybridization for MPO and by immunohistochemistry for GFP. Images taken through the Alexa-488 and TRITC channels are shown, followed by a merged image. In all GFP-positive cells (green), some MPO in situ signal (red) can be seen. Examination of individual sections confirms colocalization of these markers. (D) Summed Z stacks of consecutive images through the ICM of a day 3 transgenic zebrafish stained by in situ hybridization for L-plastin and by immunohistochemistry for GFP. Images taken through the Alexa-488 and TRITC channels are shown, followed by a merged image. GFP-positive cells (green) do not coexpress L-plastin (red). Examination of individual sections confirms absence of colocalization of these markers. (E) Tailfins of anesthetized wild-type AB zebrafish were transected at 4 days after fertilization. At the time points indicated, fish were anesthetized, then fixed and stained as described in “Materials and methods.” The number of neutrophils participating in the inflammatory response (excluding cells in the posterior blood island) was assessed. Data shown are mean ± SEM, n = 3, with 5 replicates per experiment. (F) Tailfins of anesthetized transgenic zebrafish were transected at 4 days after fertilization. At the time points indicated, individual fish were anesthetized and imaged as described. The number of fluorescent neutrophils participating in the inflammatory response was assessed. Data shown are mean ± SEM (n = 5). (G) Images taken for the counts shown in panel E were processed (IPlab) to quantify the total area of fluorescence. For each image, a series of fluorescence images in Z were projected into a single image using the maximum intensity at each point. These images were normalized, and a region of interest was defined corresponding to the area of inflammation quantitated. Segmentation of this region was performed in IPlab, and the area of fluorescence was exported to Excel. The procedure was automated by use of an IPlab script. (H) Fluorescence counts of individual larvae at 6 and 24 hours after injury (hpi) are shown. The individual pattern of resolution is evident for each fish. (I) Images showing a single transgenic zebrafish larva at the time points indicated (hpi). The accumulation of fluorescent cells and their subsequent removal can be clearly seen.

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