Immunohistochemical-stained slides were digitized using a Pannoramic 250 Flash digital microscope (P250 Flash, 3DHISTECH). Whole slides were scanned using brightfield scan mode with a 20X/NA0.80 Zeiss Plan-Apochromat dry objective (Zeiss) (Oberkochen, Germany), and images were acquired with a two megapixel 3CCD color camera (CIS Cam Ref #VCC-FC60FR19CL, CIS Americas Inc., Tokyo, Japan), achieving a 0.39 μm/pixel resolution. Pannoramic Viewer and HistoQuant software were used for viewing and analyzing the digital slides, respectively (RTM 1.15.3, 3DHISTECH). A minimum of 8 annotations per slide covering 50% of the entire tissue were analyzed using the same profile with the following characteristics: noise reduction (median filter strength = 0), object definition (HSV: 137 < Hue < 226, 26 < Saturation < 189, 17 < Value < 41), no filtering by size and no object separation. These settings allowed the automatic segmentation of the detected objects and the measurement of the number of detected objects per mm2.
Pannoramic 250 flash digital microscope
Manufactured by 3DHISTECH
Sourced in Hungary
The Pannoramic 250 Flash is a digital microscope designed for high-throughput whole slide imaging. It features a high-speed image acquisition system and is capable of scanning glass slides at a resolution of up to 0.2 microns per pixel.
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3 protocols using pannoramic 250 flash digital microscope
1
Digital Slide Analysis for Immunohistochemical Quantification
2
Immunohistochemical Evaluation of LOX Family
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Protein expression of LOX family members was detected using immunohistochemistry assays with antibodies against LOX (NB100-2527, 1:300, Novus Biologicals, Littleton, CO, USA), LOXL1 (sc-66949, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), LOXL2 (sc-48723, 1:100, Santa Cruz Biotechnology), LOXL3 (37906, 1:300, US Biological, Swampscott, MA, USA), and LOXL4 (ALX-215-067-R050, 1:100, Enzo Life Sciences, Farmingdale, NY, USA). Collagen expression was evaluated by Masson’s trichrome staining. The experiments were repeated with at least three different samples from each group. Image acquisition was performed using a Pannoramic 250 Flash digital microscope (3DHISTECH, Budapest, Hungary).
3
Histological Analysis of Zebrafish Muscle
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Whole-fish transverse sections were cut at the vent level. The frozen slices were made and stained with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA, GeneTex) as our previous report (19) . Images of transverse sections of muscle tissue and muscle fibres were obtained by scanning the slices using a Pannoramic 250 Flash digital microscope (3DHISTECH). In fish, white muscle is the predominant component, accounting for > 70 % of mass, of skeletal muscle (42) . Thus, the histological characterisation of zebrafish skeletal muscle in this study mainly focused on white muscle. The total cross-sectional area of white muscle and the individual equivalent mean diameter of white muscle fibres from the whole-fish transverse section were measured as previously described (21) .
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