Image acquisition of stained IHC biopsy sections was performed by a ZEISS Axiocam 105 colour Digital Camera and a 10× or 20× objective. The ZEISS Axiocam 105 colour is a compact 5 megapixel camera (2560 × 1920 pixels) for high‐resolution images with a 1/2.5” sensor. The images were stored in CZI format. The images were analysed with the cell image analysis software CellProfiler (Broad Institute).24 In CellProfiler different algorithms for image analysis are available as individual modules that can be modified and placed in sequential order to form a pipeline that can be used to identify and measure biological objects and features in images. Pipelines for Ki‐67, loricrin, filaggrin and pan‐LCE3 analysis were created (available on request). Data visualization and statistical analysis were performed using Instant Clue Software.25
Axiocam 105
Manufactured by Zeiss
Sourced in Germany, United States, Switzerland
The Axiocam 105 is a high-performance digital camera designed for microscopy applications. It features a 5-megapixel CMOS sensor and delivers fast image acquisition at 15 frames per second. The camera is capable of capturing high-quality images with a wide dynamic range and low noise levels.
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Lab products found in correlation
Primovert, by Zeiss (8 mentions)
Stemi 305, by Zeiss (7 mentions)
Stemi 508, by Zeiss (6 mentions)
Primovert microscope, by Zeiss (6 mentions)
Axio imager, by Zeiss (6 mentions)
Primovert bright field microscope, by Zeiss (5 mentions)
Stemi 508 stereo microscope, by Zeiss (4 mentions)
Axio lab a1 microscope, by Zeiss (4 mentions)
Axio zoom v16, by Zeiss (4 mentions)
Zen 2, by Zeiss (4 mentions)
Axioplan microscope, by Zeiss (4 mentions)
Primo star, by Zeiss (3 mentions)
Zen core, by Zeiss (3 mentions)
Axio lab a1, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Axioscope a1 light microscope, by Zeiss (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Primo star microscope, by Zeiss (2 mentions)
Stemi 2000 c, by Zeiss (2 mentions)
135 protocols using axiocam 105
1
Automated Quantitative Analysis of IHC Biopsy Images
2
Quantifying Epidermal Protein Expression
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Image acquisition of HEE immunostainings was performed by a ZEISS Axio Imager equipped with a ZEISS Axiocam 105 color Digital Camera and a 40× objective. The ZEISS Axiocam 105 color is a compact 5-megapixel camera (2560 × 1920 pixels) for high resolution images with a 1/2.5” sensor. Two images per slide were chosen as representative for the whole culture and stored in CZI format. The images were analyzed with the cell image analysis software CellProfiler (Broad Institute) [64 (link)]. Software pipelines for filaggrin (FLG), loricrin (LOR), and involucrin (IVL) analysis were created (available upon request) and GraphPad Prism 9.0 was used for the visualization of the data. The quantified data is shown as the protein area occupied (µm2) per millimeter length of the epidermis. Because this value does not normalize for the thickness of the epidermis (differences in epidermal thickness were observed after compound stimulation), we also calculated the area of expression as percentage of the epidermal surface for comparison.
3
Imaging and Analysis of Immunohistochemical-Stained HEE
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Image acquisition of immunohistochemical-stained HEE tissue sections was performed by a ZEISS Axio Imager equipped with a ZEISS Axiocam 105 color Digital Camera (Zeiss, Oberkochen, Germany) and a ×10 or ×20 objective. The ZEISS Axiocam 105 color is a compact 5-megapixel camera (2,560 ×1,920 pixels) for high-resolution images with a 1/2.5” sensor. The images were chosen as representative of the whole culture and stored in CZI format. The number of fields ranges from one to three images per slide (depending on the variation of proliferation in the HEE). The images were analyzed with the cell image analysis software CellProfiler (Broad Institute, Cambridge, MA) (McQuin et al., 2018 ). In CellProfiler, different algorithms are available that can be modified and placed in sequential order to form a pipeline for image analysis. Pipelines for Ki-67 and EdU analysis were created (available on request). Data visualization and statistical analysis were performed using GraphPad Prism (GraphPad software, San Diego, CA). To determine statistical significance between multiple groups (n ≥ 3 groups), the Kruskal‒Wallis test (a ranked-based nonparametric test) was used for all data obtained with CellProfiler. If significant, Dunn’s multiple comparison posthoc test was performed (∗P < 0.05). In each HEE culture experiment, the human KC donors used are biological replicates.
4
Quantitative Analysis of Cell Morphology
5
Porcine Intestine Adhesive Interactions
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After obtaining freshly collected porcine intestine from Schlachtbetrieb St. Gallen, Switzerland, in situ, ex situ and Tachosil adhesives were applied on the exterior surface of the fresh porcine intestine (not perforated). After application the treated intestine was sampled using an (8 mm) biopsy punch. The collected biopsies were immediately placed in a 4% formalin solution. After 24 h in formalin, the samples were separated from the bulk of the swollen hydrogel adhesive, leaving the interface intact, using surgical scissors. The same procedure was followed in the context of a stationary, post-surgery simulating experiment, where the as prepared samples were brought in contact with SIF at 37 °C, 100% humidity and light agitation. Intestinal tissue was subjected to both pancreatin rich and depleted SIF as a control.
The collected biopsies were then sent to Sophistolab AG, Muttenz, Switzerland where they were block paraffinized, cut and stained using H&E. The samples were incubated for 5 min in Hematoxylin and 30 s in Eosin solutions. For preservation, the samples were dehydrated in steps (successively 70%, 80%, 90%, 96%, 100% EtOH, 2% isopropanol, 2 × xylene, 2 min each) and mounted for subsequent storage. A ZEISS Primovert Microscope with an Axiocam 105 (Zeiss, Feldbach, Switzerland) color camera was used for image acquisition.
The collected biopsies were then sent to Sophistolab AG, Muttenz, Switzerland where they were block paraffinized, cut and stained using H&E. The samples were incubated for 5 min in Hematoxylin and 30 s in Eosin solutions. For preservation, the samples were dehydrated in steps (successively 70%, 80%, 90%, 96%, 100% EtOH, 2% isopropanol, 2 × xylene, 2 min each) and mounted for subsequent storage. A ZEISS Primovert Microscope with an Axiocam 105 (Zeiss, Feldbach, Switzerland) color camera was used for image acquisition.
6
GUS Staining of Transgenic Plant Tissues
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Seeds dissected out of the ovary, longitudinally bisected receptacle fruits, leaves, and open flowers from T0 generation transgenic plants were stored in 100 mM sodium phosphate buffer (pH 7.4) during the harvesting process. Next, sodium phosphate buffer was removed and GUS staining solution was added (100 mM sodium phosphate buffer, pH 7.4, 1 mg/mL X-glucuronic acid, 0.5 mM potassium ferricyanide, and 0.5 mM potassium ferrocyanide). Tissue was vacuum infiltrated for 30 min and then incubated overnight at 37 °C. Stained tissues were passed through an ethanol series (20%, 35%, 50%) followed by 30 min of incubation in FAA (50% ethanol, 5% formaldehyde, 10% acetic acid, water to volume). Tissues were stored in a final solution of 70% ethanol, observed using a stereo microscope, and photographed using a Zeiss Axiocam 105 color camera. The staining procedure was adapted based on published protocols53 (link),54 (link). At least 3 fruits from each developmental stage from each of at least 3 independent transgenic lines were GUS stained for all constructs. Representative photos are shown.
7
Histological Analysis of Liver and Lungs
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Histological analyzes were performed with snap-frozen tissue. Liver and lungs were fixed in formaldehyde, and clarification, dehydration and inclusion in paraffin were carried out. The compounded paraffin-blocks were cut in sections with a thickness of 5 µm by a microtome (Microm HM400). Sections of hydrated and deparaffinised tissues were stained with hematoxylin and eosin (HE). After the staining procedures, images (magnification 25x, 100x, 400x) were obtained with a Carl Zeiss microscope used for this purpose and a AxioCam 105 Colour camera with the software Zen 2012 (blue edition, Carl Zeiss) was used for image capturing. Trichrome staining was performed according to Masson’s.
8
Cytotoxic Effects of Stx2a-subunits on Cell Lines
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To investigate cytotoxic effects of the Stx2a-subunits on HeLa, Vero B4, and HCT-116 cells, Nunc® permanox 8-well chambers (Thermo Fisher Scientific, Waltham, MA, USA) were used. Cell cultures were grown and passaged as described above. For analysis, 1.0 × 104 cells/well were seeded in the 8-well chamber at a total volume of 400 µL/well. Cells were incubated at 37 °C, 5.0% CO2 for 24 h. For intoxication, each well was washed with 400 µL DPBS and cell type-dependent fresh medium was added to the cells. Cells were incubated with 0.5 µg/mL total protein concentration and incubated further at 37 °C, 5.0% CO2. After 24 and 48 h cells were analyzed by microscopy using an inverted microscope Axio Vert.A1 (Zeiss, Oberkochen, Germany) equipped with a color camera (Axiocam 105, Zeiss, Oberkochen, Germany). Pictures were detected using a 40×/1.25 objective (Zeiss, Oberkochen, Germany) and processed by the program ZEN light (Zeiss, Oberkochen, Germany). Experiments were conducted as two technical replicates in at least three biological replicates.
9
In situ Hybridization of LsHSCARB mRNA in Salmon Louse
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The localization of LsHSCARB mRNA was detected in the adult female salmon louse by using in situ hybridization (ISH) as earlier described57 (link),58 (link). An RNA antisense (AS) probe of 731 bp was made from a target specific cDNA template (see Table 1 for primer sequences). A sense (S) probe acted as negative control for the transcript localization, whereas hybridization with a known set of probes detecting LsTryp1 acted as positive control59 (link). The labelled probes were visualized by using anti-digoxigenin (DIG) alkaline phosphatase fragment antigen binding (FAB) fragment (Roche) and a chromogen substrate containing levamisole (Sigma), nitroblue tetrazolium (NTB; Roche) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Roche). Microscopy images were captured by an Axio Scope A1 light microscope connected to Axiocam 105 (Zeiss).
10
Intravital Microscopy of Wound Healing
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Prior to each intra-vital examination, mice were anesthetized by isoflurane (Baxter, Opfikon, Germany) anesthesia, 2% for initializing anesthesia and 0.25% for maintenance of anesthesia.
Microscopic pictures of the wound in the dorsal skinfold chamber were registered with AxioCam 105 color from Zeiss (Carl Zeiss microscopy, Jena, Germany) at day 0, 3 and 6 after operation. Densitometric measurements of the wound were assessed with Image J [17 (link)].
Microscopic pictures of the wound in the dorsal skinfold chamber were registered with AxioCam 105 color from Zeiss (Carl Zeiss microscopy, Jena, Germany) at day 0, 3 and 6 after operation. Densitometric measurements of the wound were assessed with Image J [17 (link)].
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