Evaluation of the sections was carried out with a computerized research microscope (LEICA DM RXE, Leica Microsystems AG). Slides were assessed quantitatively with a computerized digital camera analysing the mean colour intensity (i.e. greyscale values) of cells or tissues (analysis pro ® – version 5.0; Soft Imaging System GmbH, Muenster, Germany). This procedure was always performed under standardized conditions, thus eliminating primarily errors normally occurring during visual grading of histochemical colour intensities. Digitalized pictures were taken, white balance was performed (i.e. measurement in a tissue-free district of the section thus representing 100% transmission), randomly selected regions of interest were defined containing immunopositive structures, and colour intensity was measured. After assessing seven to nine tissue areas within the tissue, for each immunopositive structure per slide, the mean value per pixel per structure and animal was calculated. Greyscale values (GS or GSV) were transformed into extinction (E) which is proportional to dye concentrations at the level of the sections [E = Lg1(GS/256)−1 ] (Schroeter-Vogt, 2011 ), and then, this value was used for further statistical analysis.
Dmrxe
Manufactured by Leica
Sourced in Germany
The DMRXE is a high-performance research microscope from Leica Microsystems. It is designed for advanced applications in scientific research and analysis. The DMRXE offers superior optical quality, stability, and versatility, making it a valuable tool for a wide range of microscopy-based studies.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph image analysis program, by Leica (2 mentions)
Dfc490, by Leica (2 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Technovit 9100 new, by Kulzer (1 mentions)
Microtome, by Leica (1 mentions)
Vectashield containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 confocal scanner, by Leica (1 mentions)
Sp1600, by Leica (1 mentions)
Hct software, by Hamamatsu Photonics (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
7 protocols using dmrxe
1
Quantitative Analysis of Immunopositive Structures
2
Quantifying Intestinal Epithelial VDR Levels
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All immunohistochemically stained tissue sections were assessed and evaluated under the microscope at 400× magnification (Leica DM RXE, Leica Microsystems). To conduct a semi-quantitative assessment of VDR concentrations in the surface epithelium of all intestinal sections, each of the three sections were tested in 250–350 nuclei in different fields. From duodenum (DD), ileum (IL) and colon (CO) – on the surface epithelium from the base to the tip of the villi, three fictive lines were taken by separating the villi in three parts: base/bottom, middle and the tip.
Dependent on the grade of the brown colour, enterocyte nuclei were counted in five separate categories: negative, very weak, weak, middle-strong and strong colour. After the visual grading of staining for instead of or colour intensity (CI) – CI0 = negative; CI0.25 = very weak; CI1 = weak; CI4 = middle-strong positive; and CI9 = strong positive – and frequency of the tissues, the immunoreactive score (IRS) could be calculated by multiplying the relevant number of nuclei with an exponential conversion factor for the colour intensity (CI) and adding the individual products. The following formula was used (Liesegang et al., 2007 (link), 2008 ):
Dependent on the grade of the brown colour, enterocyte nuclei were counted in five separate categories: negative, very weak, weak, middle-strong and strong colour. After the visual grading of staining for instead of or colour intensity (CI) – CI0 = negative; CI0.25 = very weak; CI1 = weak; CI4 = middle-strong positive; and CI9 = strong positive – and frequency of the tissues, the immunoreactive score (IRS) could be calculated by multiplying the relevant number of nuclei with an exponential conversion factor for the colour intensity (CI) and adding the individual products. The following formula was used (Liesegang et al., 2007 (link), 2008 ):
3
Confocal Imaging of Chi-CLP Stabilized Emulsions
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The emulsions stabilized by 1 wt% chi-CLP (50 mg/g) were imaged with the confocal laser scanning microscope (Leica DMRXE, Germany). Emulsions were diluted 50 times with deionized water or with 6 wt% STP aqueous solution followed by staining the oil with Nile red (1 mg/mL in ethanol) (ca. 50 μl of Nile red in 1 ml emulsion) prior to measurement. For each sample, a drop (5 μl) of the Nile red-stained emulsion was placed on the glass slide for imaging at the wavelength of 488 nm, using a 10 × air objective and a 63 × oil immersion objective.
4
Histomorphometric Analysis of Vertebral Bone
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L5 was fixed in 4% buffered formalin for 1 week and then stored for a few weeks in 70% ethanol. Thereafter, L5 was embedded in Technovit 9100 New® (Heraeus Kulzer GmbH, Wehrheim, Germany) and cut longitudinally using a Leica microtome (RM 2165, Leica Instruments GmbH) to a thickness of 5 µm. The sections were deacrylated, stained with Toluidine Blue O (Merck, Darmstadt, Germany), and mounted with Eukitt (O. Kindler GmbH, Freiburg, Germany) [30 (link)]. The sections were digitalized using a digital camera (Leica DFC490) and a zoom stereo microscope (Leica DMRXE) and analyzed with the aid of the MetaMorph image analysis program (Leica, Bensheim, Germany). Three randomly chosen fields of 0.1 mm2 within the histological section were taken for the analyses. The following parameters were measured according to ASBMR nomenclature: osteoblast number per bone perimeter (N.Ob/B.Pm), osteoclast number per B.Pm (N.Oc/B.Pm), and osteocyte number per bone area (Ot/B.Ar) [24 (link), 31 (link)]. The criteria for the morphological identification of osteoblasts and osteoclasts were as follows. Cuboid-shaped cells that covered trabecular bone were counted as osteoblasts, whereas multinucleated cells that were resorbing bone were counted as active osteoclasts [32 (link)].
5
Immunolocalization of cMyc-tagged Transporters
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For immunolocalization of N-terminally cMyc-tagged TbAAT5-3 and TbAAT16-1, PCF over-expressing TbAAT5-3 (Tb427.08.4720) or TbAAT16-1 (Tb427tmp.01.7500) were fixed on poly-lysine slides with 4% (w/v) paraformaldehyde, followed by permeabilization with 0.2% (v/v) TX-100 in PBS. Antibody against cMyc (monoclonal antibody, mouse, clone 9E10, Santa Cruz Biotechnology) was applied at a dilution of 1:1000 in 5% (w/v) skim milk powder in PBS, followed by Alexa Fluor® 488, Goat anti-Mouse IgG (H+L) (Life Technologies), at a dilution of 1:1000. Coverslips were mounted with Vectashield® containing 4’,6-diamidino-2’-phenylindole (DAPI; Vector Laboratories) and images were obtained using a confocal microscope (Leica DM RXE, equipped with a Leica TCS SP2 confocal scanner) in sequential scanning mode. Alexa Fluor® 488 was excited with a wavelength of 488 nm and emission was detected at 500–600 nm. DAPI was detected at 410–610 nm after excitation at a wavelength of 405 nm. Final images were analyzed using Fiji software.
6
Histomorphometric Analysis of Tibial Bone
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The tibia samples were exposed to sequential ascending concentrations of ethanol and embedded in Technovit® 9100 medium (Heraeus Kulzer GmbH, Wehrheim, Germany). Sections 150 µm in thickness were cut transversally 2 cm distal to the knee surface using a diamond saw microtome (Leica SP 1600, Leica Instruments GmbH, Nussloch, Germany). Three sections of the tibia were mounted with Eukitt medium (O. Kindler GmbH, Freiburg, Germany), digitalized using a digital camera (Leica DFC490) and a zoom stereo microscope (Leica DMRXE), and analyzed with the aid of the MetaMorph image analysis program (Leica, Bensheim, Germany) (Figure 3 J). The following parameters were measured: the thickness of CG-stained new bone at the endosteal and periosteal sites of the cortical bone, marrow diameter (Ma.Dm), bone diameter (B.Dm), and the bone-to-marrow diameter ratio (B.Dm/Ma.Dm) [40 (link)].
7
Nocodazole Treatment and Lineage Tracing
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For nocodazole treatment, coverslips were assembled into observation chambers with medium containing 0.08 µM nocodazole and fields of cells were continuously followed by video time-lapse microscopy at 37°C for 6 h. After 6 h, the field of view was marked with a diamond scribe; the bottom of the observation chamber was removed and washed out with fresh medium several times before being reassembled with fresh medium as previously described (Uetake and Sluder, 2012 ). The previously marked fields were continuously followed for at least 96 h. Lineage tracing of individual cells was performed as previously described (Uetake et al., 2007 (link)). For IAA and SB203580 treatment, cells were exposed to the drug for the duration of imaging. Images were collected using a microscope (DMRXE; Leica) equipped with phase-contrast optics and a 10× 0.3 NA objective (Leica). Images were captured with an Orca ER (Hamamatsu Photonics) camera using HCT software (Hamamatsu Photonics) and exported as AVI movies to be viewed with QuickTime (Apple).
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