Confocal microscopy was conducted with a Leica TCS SP8 STED 3× microscope and LASX navigator software (Leica). Images were acquired using an HC PL APO CS2 63× 1.4 numerical aperture oil objective lens in combination with highly sensitive hybrid (HyD) detectors in photocounting mode. Resolution was set to a pixel size of 50 nm by 50 nm or 20 nm by 20 nm for confocal and STED imaging, respectively. After scanning, deconvolution was performed using CMLE (for confocal images) and GMLE algorithms (for STED images) in Huygens Professional software (Scientific Volume Imaging; Huygens, the Netherlands).
Las x navigator software
Manufactured by Leica
Sourced in Italy
Leica LAS X Navigator software is a powerful tool designed to streamline the management and analysis of microscope images. It provides a comprehensive interface for organizing, visualizing, and processing digital images acquired from Leica microscopes.
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Lab products found in correlation
Dm6 microscope, by Leica (30 mentions)
Masson trichrome staining, by Bio-Optica (3 mentions)
Dmi8 inverted microscope, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Masson s trichrome, by Bio-Optica (2 mentions)
Tcs sp8 sted 3 microscope, by Leica (1 mentions)
Dfc7000 t ccd camera, by Leica (1 mentions)
Microsystems sp8 confocal microscope, by Leica (1 mentions)
Rat tail collagen type 1, by Thermo Fisher Scientific (1 mentions)
4 oht tamoxifen, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Anti gfap antibody, by Santa Cruz Biotechnology (1 mentions)
Anti iba1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti cox 2 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti mpo antibody, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated bovine anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Biotin conjugated goat anti mouse igg and avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ethanol solution, by Merck Group (1 mentions)
Xylene, by Bio-Optica (1 mentions)
37 protocols using las x navigator software
1
Confocal and STED Microscopy Protocol
2
Quantifying Muscle Fiber Damage
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Fluorescently stained slides were imaged on a Leica Microsystems SP8 confocal microscope in widefield-mode. The system consisted of a Leica DMi8 inverted microscope with images acquired via a ×10 objective (HC PL APO CS2, NA 0.4) and a DFC7000T CCD camera. For each mechanically damaged and control sample, n = 6 cross-sections were imaged with 10x magnification through a FITC filter (Ex 450–490 nm, DC: 510, Em 515 nm LP), followed by a Y5 filter (Ex 590-650 nm, DC: 660, Em 662-738 nm). Full cross-sectional images were composed through automated tiling and stitching with the Leica LAS X Navigator software (Leica Application Suite X V.3.1.5.16308).
All images were imported into FIJI/ImageJ and converted to 8-bit greyscale images before an established analysis workflow was performed. In short, all images were background subtracted and normalised before further processing. For control tissue sections, the combined average intensity was determined for each sample. This value was set as threshold to mask unlabelled fibres in sections from the indented areas. Following segmentation with Cellpose44 (link) to separate individual myofibers, the number of dye-positive cells in the indented area was counted for each cross-section and averaged across each sample.
All images were imported into FIJI/ImageJ and converted to 8-bit greyscale images before an established analysis workflow was performed. In short, all images were background subtracted and normalised before further processing. For control tissue sections, the combined average intensity was determined for each sample. This value was set as threshold to mask unlabelled fibres in sections from the indented areas. Following segmentation with Cellpose44 (link) to separate individual myofibers, the number of dye-positive cells in the indented area was counted for each cross-section and averaged across each sample.
3
Angiogenesis Assay of Vegfr2 Mutant Embryos
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Vegfr2Y1212F/+ C57Bl/6: R26StopFLMYC allele mice and Vegfr2Y1212F/+ C57Bl/6: Cdh5‐CreER mice were put together for timed breeding. At E9.5, 100 μl of 4OHT‐tamoxifen (Sigma, H7904) (10 mg/ml dissolved in peanut oil) was injected intraperitoneally into pregnant females. At E11.5, embryos were harvested and stored on ice in PBS until prepared for collagen embedding by dissecting away the head and the tail of each embryo. Genotyping of the embryos was done using the embryonic tailpiece. The embryos were embedded between two layers of rat tail type I collagen (Life technologies) and cultured in DMEM supplemented with 15% FCS and 30 ng/ml VEGFA for 10 days. Samples were fixed in 4% PFA and analyzed by microscopy after immunostaining with antibodies. Tile scan z‐stack images were taken with a Leica TCS SP8 Confocal Microscope with PMT and HyD detectors and LAS X Navigator software (Leica). Image analysis was done using ImageJ software (NIH) on a 2,000 × 2,000 μm region of the sprouting front of each explant.
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Vegfr2Y1212F/+ C57Bl/6: R26StopFLMYC allele mice and Vegfr2Y1212F/+ C57Bl/6: Cdh5‐CreER mice were put together for timed breeding. At E9.5, 100 μl of 4OHT‐tamoxifen (Sigma, H7904) (10 mg/ml dissolved in peanut oil) was injected intraperitoneally into pregnant females. At E11.5, embryos were harvested and stored on ice in PBS until prepared for collagen embedding by dissecting away the head and the tail of each embryo. Genotyping of the embryos was done using the embryonic tailpiece. The embryos were embedded between two layers of rat tail type I collagen (Life technologies) and cultured in DMEM supplemented with 15% FCS and 30 ng/ml VEGFA for 10 days. Samples were fixed in 4% PFA and analyzed by microscopy after immunostaining with antibodies. Tile scan z‐stack images were taken with a Leica TCS SP8 Confocal Microscope with PMT and HyD detectors and LAS X Navigator software (Leica). Image analysis was done using ImageJ software (NIH) on a 2,000 × 2,000 μm region of the sprouting front of each explant.
4
Immunohistochemical Analysis of Brain Markers
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Immunohistochemical analysis was performed as already described [50 (link)]. Subsequently, the sections were incubated overnight with an anti-Bromodeoxyuridine (BrdU) antibody (1:100; Santa Cruz Biotechnology) or anti-MPO antibody (1:250; Santa Cruz Biotechnology) or anti-NRLP3 antibody (1:250; Santa Cruz Biotechnology) or anti-COX-2 antibody (1:250; Santa Cruz Biotechnology) or anti-Iba-1 antibody (1:250; Santa Cruz Biotechnology) or anti-GFAP antibody (1:450; Santa Cruz Biotechnology). Sections were washed with PBS and incubated with peroxidase-conjugated bovine anti-mouse IgG, secondary antibody (1:2000 Jackson Immuno Research, West Grove, PA, USA). Specific labeling was provided with a biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Images were collected using a Leica DM6 microscope associated with Leica LAS X Navigator software. The number of positive cells was counted in three sections per animal and presented as the number of positive cells per high-power field.
5
Multi-marker Immunofluorescent Staining Protocol
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Immunofluorescent staining was performed on paraffin embedded samples prepared as described above. Antigen retrieval was performed in either sodium citrate or Tris-EDTA buffers followed by overnight incubation with primary antibodies against insulin (1:100; Santa Cruz), CD3 (1:100; Abcam), and PD-1 (1:50; Abcam) all co-stained with DAPI (1:3000; Thermo Fisher Scientific). The following day, slides were incubated with Alexa Fluor 488 conjugated donkey anti- rabbit secondary antibody (Invitrogen). Samples were imaged using a Leica DMi8 inverted microscope (Leica) and LAS X Navigator software (Leica).
6
Ultrastructural Analysis of Cerebral Organoids
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Cerebral organoids cultured in the presence or absence of the in vitro generated BBB for 4 days were fixed in 2,5% buffered glutaraldehyde, post-fixed in 1% Osmium tetroxide buffered solution, and embedded in epoxy resin (Embed 812 kit for Electron Microscopy, EMS Company) using standard preparative procedure for ultrastructural analyses of tissues and cells. Semithin resin sections were stained with toluidine blue and analyzed at light microscope. Then, thin sections from selected areas were stained (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted July 22, 2022. ; https://doi.org/10.1101/2022.07.22.501119 doi: bioRxiv preprint with uranyl acetate/lead citrate and examined by EM-109 ZEISS and CCD-Megaview G2 (I-TEM imaging platform software).
Image of each organoid was reconstructed using Leica LAS X Navigator Software.
The copyright holder for this preprint this version posted July 22, 2022. ; https://doi.org/10.1101/2022.07.22.501119 doi: bioRxiv preprint with uranyl acetate/lead citrate and examined by EM-109 ZEISS and CCD-Megaview G2 (I-TEM imaging platform software).
Image of each organoid was reconstructed using Leica LAS X Navigator Software.
7
Histopathological Examination of Lung Tissue
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Lung tissue sections (7 μm) were stained with hematoxylin/eosin (H/E) and phosphotungstic acid hematoxylin (PTAH) for histopathological examination. Sections were examined using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy) [61 (link)]. Every piece was viewed at a magnification of 20× for H/E and 40× for PTAH, and morphological changes were evaluated by two blinded investigators. The grading scale to score histopathologic findings was determined as previously described [62 (link)].
8
Toluidine Blue Staining of Lung Tissue
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At the end of experiment, for identification of MCs, lung tissue sections (7 μm) were stained with toluidine blue, as described previously [63 (link)]. Every section was observed at a magnification of 100×, using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy).
9
Histological Evaluation of Tibiofemoral Joints
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The MIA- and vehicle-injected tibiofemoral joints were dissected immediately after sacrifice and post-fixed in neutral buffered formalin (containing 4% formaldehyde) as previously described [31 (link)]. Mid-coronal tissue sections (5 μm) were stained with H/E, observed using a Leica DM6 microscope at × 10 magnification (Leica Microsystems SpA, Milan, Italy) equipped with a motorized stage and associated with Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy). Collagen content was assayed according to the manufacturer’s protocol (Bio-Optica, Italy, Milan), with tissue sections being stained with Masson’s trichrome. Proteoglycan depletion and cartilage destruction were assayed using Safranin O/Fast green staining. The number and degranulation extent of mast cells were also assessed in tissue sections stained with toluidine blue as previously described [36 (link)].
Finally, a modified Mankin histologic scoring system was used to evaluate cartilage damage, from 0 (normal histology) to 12 (complete disorganization and hypocellularity) [37 (link)]. The photographs obtained (n = 5 photos from five slides for each sample) were collected from all animals in each experimental group. The histological coloration (five slides for each same sample) was repeated three times on different days.
Finally, a modified Mankin histologic scoring system was used to evaluate cartilage damage, from 0 (normal histology) to 12 (complete disorganization and hypocellularity) [37 (link)]. The photographs obtained (n = 5 photos from five slides for each sample) were collected from all animals in each experimental group. The histological coloration (five slides for each same sample) was repeated three times on different days.
10
Lung Fibrosis Scoring and Mast Cell Analysis
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Lung tissues samples were collected 7 days from bleomycin injection. Tissues were fixed in buffered formaldehyde solution (10% in PBS), histological sections were stained with haematoxylin and eosin and Masson Trichrome and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) associated with Leica LAS X Navigator software (Leica Microsystems SpA). The severity of lung fibrosis was scored on a scale from 0 to 8 as already published [19 (link)]. Lung sections were stained with toluidine blue to enumerate mast cells [35 (link)].
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