Immunofluorescent staining of human lung cancer tissue sections was performed as described elsewhere54 (link). Bright field and fluorescence Images were acquired with a 20X (0.75 NA) objective on a Nikon A1+ confocal or with a 10X (0.45 NA) objective on a Nikon Eclipse Ti inverted widefield microscope (Nikon Corporation, Tokyo, Japan) equipped with DS-Qi2 monochrome camera (fluorescence) and DS-Ri2 color camera (color Brightfield) using NIS Elements software (Nikon). Raw images were deconvolved with Huygens Professional version 15.05 (Scientific Volume Imaging, The Netherlands, https://svi.nl/ ) and processed in ImageJ (NIH, Bethesda, https://imagej.nih.gov/ij ). Briefly, deconvolved images were thresholded, smoothened and watershed segmented before creating a mask for DAPI channel to score for total number of cells per field. Other channels were processed identically and multiplied with the nuclear mask before scoring for number of single positive cells. Double positive cells were identified using RG2B colocalization plugin. Scores were expressed as percentage.
Huygens professional version 15
Manufactured by Scientific Volume Imaging
Huygens Professional version 15.05 is a software tool for the analysis and processing of microscopy images. It provides advanced algorithms for deconvolution, segmentation, and visualization of 3D and 4D microscopy data.
Automatically generated - may contain errors
2 protocols using huygens professional version 15
1
Immunofluorescent Staining of Lung Cancer Tissue
2
Confocal Microscopy of Protein Gelation
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All samples were confined to capillaries with a square cross-section of 0.50 × 0.50 mm (Vitocrom) and sealed on the ends with Norland 61 optical adhesive. Confocal laser scanning microscopy Leica TCS with a white light laser emitting at 500 nm was used to study any gelation, using a NA 1.4 63× oil immersion objective, with a resolution of 0.1 μm in x and y and 0.6 μm in z. The channels used for the proteins were 488 nm for eGFP and 587 nm for native and cationised mCherry. Scans of the capillary in the z-axis were also acquired to analyse the network structure in 3d, where care was taken to assure the pixel size was equal in all axes (100 nm per pixel). Images of the binary networks were deconvolved with Huygens Professional version 15.05 (Scientific Volume Imaging, The Netherlands, http://svi.nl ).
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