Multichannel images were acquired using a Nikon A1R Ti:E inverted confocal microscope with 1AU pinhole dimension, using a 40× Plan Apo/numerical aperture (NA) 1.25 oil objective (histological staining) or 10× Plan Flour/NA 0.3 (GCaMP6f/DAPI acquisition). Sequential acquisition of the four channels was performed with the laser lines DAPI 402 nm, GCaMP6f 488 nm, NeuN/Alexa555 562 nm, GAD67/Alexa647 639 nm, and 450/50; and with 525/50 and 595/50 filter cubes. Histological sections stained with DAPI were images with a Leica DMR upright microscope with Retiga 2000R camera using an epifluorescence mercury lamp and PL FLUOTAR 10×/0.30 and PL FLUOTAR 20×/0.50 objectives.
Dmr upright microscope
Manufactured by Leica
Sourced in Germany
The Leica DMR upright microscope is a high-performance laboratory instrument designed for a variety of applications. It features a robust and ergonomic design, with a range of optical configurations to suit different research needs. The DMR provides reliable and precise imaging capabilities for users in various scientific fields.
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Lab products found in correlation
Xylene, by Avantor (3 mentions)
Periodic acid schiff, by Merck Group (3 mentions)
Image pro plus 7, by Media Cybernetics (2 mentions)
Progres c14, by Jenoptik (1 mentions)
Paraffin, by Avantor (1 mentions)
Absolute ethanol, by Avantor (1 mentions)
Eosin y, by Avantor (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Hematoxylin, by Ampliqon (1 mentions)
Nis element, by Nikon (1 mentions)
Paraformaldehyde (pfa), by CellPath (1 mentions)
9 protocols using dmr upright microscope
1
Multichannel Confocal Microscopy Imaging
2
Tissue Fixation and Immunohistochemical Analysis
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All protocols were approved by the Sinai Health System Research Ethics Boards. Tissue samples were fixed in 10% formalin for 24 h at room temperature. Serial sectioning was performed at three levels, 50 mm apart. Each tissue edge was stained red with Tissue-Marking dye to orientate the specimen for paraffin embedding (Fig. S1 a). The strips of peritoneal tissue were paraffin embedded by lining up the three strips parallel to each other, with the mesothelial layer oriented to one side (Fig. S1 b). Sections (5 μm) were stained with hematoxylin and eosin (H&E) according to standard protocols using the Veristain Gemini Automated Slide Stainer (Thermo Scientific) at the University of Toronto Dentistry Pathology Core, and examined with a Leica DMR upright microscope. For IHC staining, slides were stained with CDX2 1:50 or D2-40 1:50 antibodies using the fully automated Dako Omnis platform (deparaffinization and retrieval built in), using the Dako OMNIS detection kit (Ref: GV800). Slides were exposed to high pH for 15 min, antibody incubation for 10 min, polymer detection for 20 min, substrate chromogen for 5 min and finally Dako hematoxylin for 3 min. This IHC processing was performed at the Department of Pathology at the Hospital for Sick Children, Toronto.
3
Luxol Fast Blue and Eosin Staining of Spinal Cord
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Luxol fast blue and eosin staining was used to visualise grey and white matter and lesion size in the spinal cord tissue sections. The sections were incubated at in 0.1% Luxol fast blue at 60°C for six hours then differentiated in 0.1% lithium carbonate for 30 seconds before changing to 70% ethanol for 30 seconds and then rinsed off in water. The sections were counterstained with eosin for 30 seconds before being cleared with xylene and mounted. A Leica DMR upright microscope was used to capture 2.5x magnification images of the stained sections. ImageJ software was used to trace the regions of white matter, grey matter and lesion size for every section. This staining was performed twice and the values were averaged to minimise variation in the data.
4
Quantifying Rab5 Puncta in Drosophila Wing
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Fluorescent images were collected on an Olympus FV1000 confocal. Bright-field adult wing and notum photographs were taken using the ProgResC14 camera system from Jenoptik on a Leica DMR upright microscope. Images were processed in ImageJ and figures constructed using Adobe Photoshop.
For Rab5 puncta analysis, a Z stack of 0.15 μm slices at 60× magnification with 3× zoom (0.069 μm per pixel) was obtained and slices were maximally projected in ImageJ as follows. Apical was defined as the three most apical in focus slices (roughly 0.5 μm thickness), whereas subapical was defined as the next 0.5 μm (slices 4–6). To get a value for total Rab5 (Fig. 4 G), the top 10 slices were projected. Projected images were thresholded in ImageJ using the Triangle function and particles were analysed for their number, average size and total area in identically sized wild-type and mutant areas of the wing. T-tests were used to calculate statistical significance. Mutant values were normalised against wild-type values within each wing to make graphs. Data was compiled and statistically analysed using the Microsoft Excel.
For Rab5 puncta analysis, a Z stack of 0.15 μm slices at 60× magnification with 3× zoom (0.069 μm per pixel) was obtained and slices were maximally projected in ImageJ as follows. Apical was defined as the three most apical in focus slices (roughly 0.5 μm thickness), whereas subapical was defined as the next 0.5 μm (slices 4–6). To get a value for total Rab5 (
5
Three-Dimensional Deconvolution Microscopy
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Three-dimensional stacks with a 0.2-mm step were acquired by 3D deconvolution microscopy adapted on an DMR upright microscope (Leica) equipped with a CoolSNAP HQ2 charge-coupled device (CCD) camera (Photometrics) and using a 100 3 Plan Apochromat HCX oil immersion objective (NA = 1.4) controlled in the z axis by a piezoelectric motor (LVDT; Physik Instrumente). For each condition, 200-300 Hoechst-stained cells were examined in 3 biologically independent experiments. Deconvolution, when applied, was performed automatically using an iterative and measured point spread function (PSF)-based algorithm method (Gold-Meinel) on batches of image stacks. Identical processing parameters and number of iterations were used. Maximum intensity projections were performed using ImageJ software.
6
Histological Analysis of Intestinal Tissues
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Fixated tissues were dehydrated in an ethanol gradient of 77%‐99% (absolute ethanol, cat. no. 83813.360, VWR), cleared using xylene (cat. no. 28973.363, VWR) and embedded in paraffin (cat. no. 2270.60.60, Hounisen, Skanderborg, Denmark). Sections of 2 μm were stained with Meyer's Hematoxylin (cat. no. AMPQ00254.0500, Ampliqon, Odense, Denmark) and Eosin Y (cat. no. 341973R, VWR) to identify eosinophils and 0.5% Toluidine Blue (TB; cat. no. 89640, Sigma‐Aldrich) in 1 M hydrochloric acid to identify mast cells, or Periodic acid‐Schiff (PAS; periodic acid: Cat. no. 1.00524.0025, Merck and Schiff′s reagent: Cat. 3952016, Sigma‐Aldrich) to identify goblet cells.34 Slides were examined using a Leica DMR upright microscope (Leica Microsystems GmbH, Wetzlar, Germany). The software ImagePro Plus 7.0 (MediaCybernetics, Rockville, MD, US) was used for image analysis. Villus length was measured from the villus tip to the crypt‐villus junction. Cell count and villi length in the SI were averaged from three sections of three similar consecutive villi and crypts. Cell count in the colon was averaged from six individual crypts. Analysis of histological sections was performed blinded.
7
Intestinal Tissue Histomorphometry Protocol
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After overnight fixation in 4% formalin (CellPath, Newtown, United Kingdom), sections of small intestine and colon were dehydrated in a graded ethanol series from 77 to 99% ethanol (VWR Chemicals, Radnor, PS, United States). Xylene (VWR Chemicals) was used as clearing agent to replace the ethanol before the tissues were embedded in paraffin (Hounisen, Skanderborg, Denmark). Histological sections of 2 μm were stained with Hematoxylin (Ampliqon, Odense, Denmark) and Eosin (Merck, Darmstadt, Germany) to identify eosinophils, and Periodic acid–Schiff (PAS) (Merck) to identify goblet cells. The slides were examined using a Leica DMR upright microscope (Leica Microsystems GmbH, Wetzlar, Germany) and the software ImagePro Plus 7.0 (Media Cybernetics, Rockville, MD, United States) was used for images and measurements. Villus length was measured from the villus tip to the crypt-villus junction and in colon crypt depth was measured, with three villi or crypts measured/counted per animal. Analyses of histological sections were performed blinded.
8
Bacterial Microscopic Examination
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Each Gram stained section was examined using bright field light microscopy with a Leica DMR upright microscope (Leica Microsystems, Wetzlar, Germany). Photographs were taken with a Nikon Digital Sight DS-5Mc-U1 cooled colour camera (Nikon, Tokyo, Japan), using Nikon NIS Elements for Image Acquisition software (Nikon, Tokyo, Japan). All sections were screened at 10X magnification and examined for the presence of bacterial microcolonies.
9
Quantitative Histological Analysis of Intestinal Goblet Cells
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To evaluate histological changes, 1 cm of the small intestine, 38 cm distal from the stomach, and 1 cm of the large intestine, 1 cm distal from cecum, were collected (from 18 rats/group) and fixed overnight in 4% (w/v) paraformaldehyde (CellPath, Newtown, UK). Sections were dehydrated in a 77% to 99% graded ethanol series. Xylene (VWR Chemicals, Radnorm PS, US) was used as clearing agent to replace ethanol before embedding the tissue in paraffin (Hounisen, Skanderborg, Denmark). Sections of 3 µm were stained with Periodic acid-Schiff (PAS) (Merck) to identify goblet cells. Sections were examined using Leica DMR upright microscope (Leica Microsystems GmbH, Wetzlar, Germany). Goblet cells were counted from villus to crypt in two connected villi at two different places in the section of the small intestine, and at three different places in the section for the large intestine. Analysis of the sections were performed in a blinded manner.
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