Microscopy:Article Title: Whole-slide imaging and a Fiji-based image analysis workflow of immunohistochemistry staining of pancreatic islets
Article Snippet: Protocol name , Whole-slide imaging and image analysis workflow of pancreas sections.. .. Reagents/tools , Microscope • MetaSystems VSlide slide-scanner (MetaSystems, Altlussheim, Germany. This system consisted of: ▪ Microscope: Zeiss AxioImager Z2 microscope (Carl Zeiss Microscopy, Jena, Germany). ▪ Camera: monochrome CoolCube 1m camera (MetaSystems, Altlussheim, Germany). ▪ Stage: motorised five-position scanning stage (Marzhauser, Wetzlar, Germany). ▪ SlideFeeder x80 (MetaSystems, Altlussheim, Germany). ▪ A quad filter (filter set 81 HE; Carl Zeiss Microscopy, Jena, Germany) in conjunction with a Colibri.2 (Carl Zeiss Microscopy, Jena, Germany) light source to visualise Alexa Fluor 488, Alexa Fluor 647, and DAPI fluorophores. ▪ A filter specific to Alexa Fluor 594 (filter set 45 HQ TexasRed; Carl Zeiss Microscopy, Jena, Germany) in conjunction with an X-Cite 120PC Q (Excelitas Technologies, Waltham, Massachusetts, USA) light source to visualise the Alexa Fluor 594 fluorophore. Software: • Metafer software (version 3.12.6; MetaSystems, Altlussheim, Germany) to control light sources, filter sets, and exposure times for each assay. • VSlide software (version 1.1.107; MetaSystems, Altlussheim, Germany) to stitch raw images into one whole-slide image. • MetaViewer software (version 2.0.133; Metasystems, Altlussheim, Germany) to view stitched whole-slide images. • Fiji (version 1.53g; Fiji Is Just ImageJ, NIH, Maryland, USA) for subsequent image analysis.. .. Experimental design , Pancreata were removed from 132-133 days gestational age fetal lambs at post mortem examination. Pancreata were sectioned into two halves along the midline of the organ so that each half contained head, body, and tail regions. One half was preserved in neutral buffered 10% formalin and transferred into 70% ethanol after 72-hours for storage until processing. The other half was snap-frozen in liquid nitrogen for separate molecular biology experiments. Formalin-fixed pancreata were paraffin-embedded, and 5 μm thick sections were cut and placed onto SuperFrost plus adhesion slides (Thermo Fisher Scientific). Five tissue sections per lamb, at least 100 μm apart, underwent multiplexed immunohistochemistry (IHC) for insulin, glucagon, and somatostatin. A MetaSystems VSlide slide-scanner (MetaSystems) was used to image stained tissue sections. We acquired approximately 450-800 fields of view (FOV) per slide using a Plan Apochromat 20x/0.8 NA objective lens. Image analysis was performed on individual FOV images using Fiji (NIH, USA), an open-source image analysis software. We used the following image analysis workflow comprising a sequence of 2 macros: image cropping and file renaming (macro 01: cropping and renaming macro); image analysis, which included defining regions without tissue, identifying and measuring insulin-positive, glucagon-positive, and somatostatin-positive staining (macro 02: analysis macro); and followed by image quality control. .
Software:Article Title: Whole-slide imaging and a Fiji-based image analysis workflow of immunohistochemistry staining of pancreatic islets
Article Snippet: Protocol name , Whole-slide imaging and image analysis workflow of pancreas sections.. .. Reagents/tools , Microscope • MetaSystems VSlide slide-scanner (MetaSystems, Altlussheim, Germany. This system consisted of: ▪ Microscope: Zeiss AxioImager Z2 microscope (Carl Zeiss Microscopy, Jena, Germany). ▪ Camera: monochrome CoolCube 1m camera (MetaSystems, Altlussheim, Germany). ▪ Stage: motorised five-position scanning stage (Marzhauser, Wetzlar, Germany). ▪ SlideFeeder x80 (MetaSystems, Altlussheim, Germany). ▪ A quad filter (filter set 81 HE; Carl Zeiss Microscopy, Jena, Germany) in conjunction with a Colibri.2 (Carl Zeiss Microscopy, Jena, Germany) light source to visualise Alexa Fluor 488, Alexa Fluor 647, and DAPI fluorophores. ▪ A filter specific to Alexa Fluor 594 (filter set 45 HQ TexasRed; Carl Zeiss Microscopy, Jena, Germany) in conjunction with an X-Cite 120PC Q (Excelitas Technologies, Waltham, Massachusetts, USA) light source to visualise the Alexa Fluor 594 fluorophore. Software: • Metafer software (version 3.12.6; MetaSystems, Altlussheim, Germany) to control light sources, filter sets, and exposure times for each assay. • VSlide software (version 1.1.107; MetaSystems, Altlussheim, Germany) to stitch raw images into one whole-slide image. • MetaViewer software (version 2.0.133; Metasystems, Altlussheim, Germany) to view stitched whole-slide images. • Fiji (version 1.53g; Fiji Is Just ImageJ, NIH, Maryland, USA) for subsequent image analysis.. .. Experimental design , Pancreata were removed from 132-133 days gestational age fetal lambs at post mortem examination. Pancreata were sectioned into two halves along the midline of the organ so that each half contained head, body, and tail regions. One half was preserved in neutral buffered 10% formalin and transferred into 70% ethanol after 72-hours for storage until processing. The other half was snap-frozen in liquid nitrogen for separate molecular biology experiments. Formalin-fixed pancreata were paraffin-embedded, and 5 μm thick sections were cut and placed onto SuperFrost plus adhesion slides (Thermo Fisher Scientific). Five tissue sections per lamb, at least 100 μm apart, underwent multiplexed immunohistochemistry (IHC) for insulin, glucagon, and somatostatin. A MetaSystems VSlide slide-scanner (MetaSystems) was used to image stained tissue sections. We acquired approximately 450-800 fields of view (FOV) per slide using a Plan Apochromat 20x/0.8 NA objective lens. Image analysis was performed on individual FOV images using Fiji (NIH, USA), an open-source image analysis software. We used the following image analysis workflow comprising a sequence of 2 macros: image cropping and file renaming (macro 01: cropping and renaming macro); image analysis, which included defining regions without tissue, identifying and measuring insulin-positive, glucagon-positive, and somatostatin-positive staining (macro 02: analysis macro); and followed by image quality control. .
Control:Article Title: Whole-slide imaging and a Fiji-based image analysis workflow of immunohistochemistry staining of pancreatic islets
Article Snippet: Protocol name , Whole-slide imaging and image analysis workflow of pancreas sections.. .. Reagents/tools , Microscope • MetaSystems VSlide slide-scanner (MetaSystems, Altlussheim, Germany. This system consisted of: ▪ Microscope: Zeiss AxioImager Z2 microscope (Carl Zeiss Microscopy, Jena, Germany). ▪ Camera: monochrome CoolCube 1m camera (MetaSystems, Altlussheim, Germany). ▪ Stage: motorised five-position scanning stage (Marzhauser, Wetzlar, Germany). ▪ SlideFeeder x80 (MetaSystems, Altlussheim, Germany). ▪ A quad filter (filter set 81 HE; Carl Zeiss Microscopy, Jena, Germany) in conjunction with a Colibri.2 (Carl Zeiss Microscopy, Jena, Germany) light source to visualise Alexa Fluor 488, Alexa Fluor 647, and DAPI fluorophores. ▪ A filter specific to Alexa Fluor 594 (filter set 45 HQ TexasRed; Carl Zeiss Microscopy, Jena, Germany) in conjunction with an X-Cite 120PC Q (Excelitas Technologies, Waltham, Massachusetts, USA) light source to visualise the Alexa Fluor 594 fluorophore. Software: • Metafer software (version 3.12.6; MetaSystems, Altlussheim, Germany) to control light sources, filter sets, and exposure times for each assay. • VSlide software (version 1.1.107; MetaSystems, Altlussheim, Germany) to stitch raw images into one whole-slide image. • MetaViewer software (version 2.0.133; Metasystems, Altlussheim, Germany) to view stitched whole-slide images. • Fiji (version 1.53g; Fiji Is Just ImageJ, NIH, Maryland, USA) for subsequent image analysis.. .. Experimental design , Pancreata were removed from 132-133 days gestational age fetal lambs at post mortem examination. Pancreata were sectioned into two halves along the midline of the organ so that each half contained head, body, and tail regions. One half was preserved in neutral buffered 10% formalin and transferred into 70% ethanol after 72-hours for storage until processing. The other half was snap-frozen in liquid nitrogen for separate molecular biology experiments. Formalin-fixed pancreata were paraffin-embedded, and 5 μm thick sections were cut and placed onto SuperFrost plus adhesion slides (Thermo Fisher Scientific). Five tissue sections per lamb, at least 100 μm apart, underwent multiplexed immunohistochemistry (IHC) for insulin, glucagon, and somatostatin. A MetaSystems VSlide slide-scanner (MetaSystems) was used to image stained tissue sections. We acquired approximately 450-800 fields of view (FOV) per slide using a Plan Apochromat 20x/0.8 NA objective lens. Image analysis was performed on individual FOV images using Fiji (NIH, USA), an open-source image analysis software. We used the following image analysis workflow comprising a sequence of 2 macros: image cropping and file renaming (macro 01: cropping and renaming macro); image analysis, which included defining regions without tissue, identifying and measuring insulin-positive, glucagon-positive, and somatostatin-positive staining (macro 02: analysis macro); and followed by image quality control. .
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