Fluorescence in situ hybridization (FISH) analysis was performed for the WM coculture on 4% paraformaldehyde-fixed samples according to a procedure described previously (Moter and Gobel, 2000 (link)). Oligonucleotide probes specific for bacteria (Cy3-labeled EUB338mix probes) and archaea (fluorescein isothiocyanate (FITC)-labeled ARC915 probe) were used. The details of the probes design are available in the probeBase (Loy et al., 2003 (link)). The labeled samples were visualized using epifluorescence microscopy (Axioimager D2, ZEISS, Oberkochen, Germany) (Zhang and Lu, 2016 (link)). The brightness of fluorescence images was optimized by ImageJ (Ciniselli et al., 2015 (link)). The cells in the lag phase, early exponential phase, mid-log phase, and late exponential phase were collected and observed. For scanning electron microscopy (SEM), cells were collected by a syringe, fixed with 2.5% (wt/vol) glutaraldehyde in phosphate-buffered saline, and sequentially dehydrated with ethanol [20, 40, 60, 80, 95, and 100% (v/v) ethanol, 10 min for each step]. The dried samples were coated with platinum and imaged using SEM (Axio imager D2, ZEISS) (Zhang and Lu, 2016 (link)).
Axio imager d2
Manufactured by Zeiss
Sourced in Germany
The Axio Imager D2 is a microscope system designed for a wide range of research and industrial applications. It features a modular design, allowing for customization to meet specific needs. The core function of the Axio Imager D2 is to provide high-quality imaging and analysis capabilities for various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrm, by Zeiss (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Nanosem 430, by Thermo Fisher Scientific (3 mentions)
Fluoroshield mounting medium, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Axiocam hrm, by Zeiss (2 mentions)
Syto59, by Thermo Fisher Scientific (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Incucyte microscope, by Sartorius (2 mentions)
H 1200, by Vector Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fv1000 spd, by Olympus (2 mentions)
Y idt, by Nikon (2 mentions)
Axiocam hrc, by Zeiss (2 mentions)
Zen blue pro 2011 software package, by Zeiss (2 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Discovery v8, by Zeiss (1 mentions)
Sudan 3, by Merck Group (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
78 protocols using axio imager d2
1
Fluorescence in situ Hybridization of Microbes
2
GFP Reporter Microscopy Protocols
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Yeast cultures expressing GFP reporters were fixed at the appropriate time points as described (Bloom et al., 2018 ). Microscopy of yeast cells was conducted using ZEISS Axio Imager D2 compound fluorescent scope and Zeiss Zen software. Images were captured using GFP and DIC filters. Exposure time for FFL-Luciferase-GFP detection and Htt-Q97-GFP was 42 and 100 ms, respectively; 100 ms was used for both reporters in diploid strains. A minimum of 100 cells over three fields was counted and tabulated using ImageJ software with a “minimum displayed value” setting of 15 in order to reduce the contribution of cells whose fluorescence level was insufficient to evaluate. As a result of this setting, 10–20% of sampled cells for any given genotype were not counted (Schindelin et al., 2012 (link); Schneider et al., 2012 (link); Rueden et al., 2017 (link)). Blind counting tests were conducted to confirm the reliability of the measurements. C. elegans microscopy was conducted on a ZEISS Axio Imager D2 motorized compound fluorescence upright microscope with DIC and a Zeiss Axiocam MRm with ZEN image capture software. Images of Xenopus embryos were acquired with a Zeiss Discovery V8 dissecting microscope using an AxioCam MrC 5 camera and AxioVision software (Zeiss).
3
Histochemical Detection of Suberin and Lignin
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Suberin lamellae in root tissues were detected by staining cross-sections at 70 o C for 10 min with a saturated solution of Sudan III (Merck, Darmstadt, Germany) dissolved in ethanol/water (1:1; v/v), according to Gerlach (1984) . Suberin lamellae were recognized by orange color under bright-field illumination (Axio Imager D2, Carl Zeiss, Germany). Lignin deposition in cross-sections was detected by staining for several minutes with phloroglucinol/HCl at room temperature (Jensen 1962) . Transverse sections were mounted in a mixture with 10% (v/v) H 2 SO 4 in 75% (v/v) glycerol to prevent color fading. Lignified tissues were viewed as bright red/pink under bright-field illumination (Axio Imager D2, Carl Zeiss, Germany). Suberization and lignification of exodermis and endodermis were assessed for 3-5 roots per replicate. A scoring system similar to the one described by Enstone and Peterson (2005) was used to characterize suberin and lignin deposition in exodermis and endodermis: "0" represented no suberin or lignin staining (no detectable suberin lamella and lignin deposition); "1" to "3" represented slight to moderate suberin or lignin staining; and "4" to "5" represented strong to very strong suberin or lignin staining.
4
Quantification of TUNEL-Positive Cells
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The TUNEL assay was performed using the In situ Fluorometric TUNEL kit (11,684,795,910, Roche, Switzerland) according to the manufacturer’s instructions. Three to six random fields from each ileum samples and organoids were captured and quantified at a 200 × magnification using the ortho fluorescence microscope (Zeiss Axio Imager D2, German). The number of positive nuclei was confirmed by fluorescence microscope and measured with ImageJ software. The ratio of positive cell counts to all cells in the villi was calculated.
5
Immunofluorescence Microscopy Protocol
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For immunofluorescence microscopy, cells were either fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature or with methanol for 5 min at −20°C. Antibodies were diluted in PBS with 0.1% (v/v) Triton-X100 and 10% (v/v) horse serum. The cells were washed in PBS with 0.1% (v/v) Triton-X100 and incubated for 1 h with primary antibodies. The cells were washed again in PBS with 0.1% (v/v) Triton-X100 and incubated with appropriate secondary antibodies for another 1 h. The cells were washed again first in PBS with 0.1% (v/v) Triton-X100, then PBS, and finally in H2O. In the end, coverslips were mounted onto glass slides using Aqua-poly/Mount mounting medium (Polysciences). Samples were analyzed using widefield fluorescence microscopes (Leica DMRA2 or Zeiss AxioImager D2) or a confocal laser scanning microscope (Zeiss LSM 710 META). Unless otherwise indicated, images were obtained by confocal microscopy and processed using Zeiss LSM Image Browser and Adobe Photoshop software or Fiji (Schindelin et al., 2012 (link)).
6
Immunofluorescence Staining of Macrophages
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Paraformaldehyde-fixed BMDM, peritoneal macrophages, and J774 from each group were washed with PBS and permeabilized with 0.1% Triton X-100/PBS at room temperature for 10 min. Samples were then blocked in 5% rabbit serum in PBS for 1 h at room temperature and incubated at 4 °C overnight with the following primary antibodies at 1:200 dilutions in 5% rabbit serum: rabbit anti-Ki67 (Abcam, Cambridge, UK), mouse-anti-iNOS (Abcam, Cambridge, UK), and rabbit anti-Arg-1 (Invitrogen, Waltham, MA, USA). Samples were washed three times for 5 min with PBS. They were then incubated for 1 h at room temperature with the following secondary antibodies at 1:400 dilutions in 5% rabbit serum: anti-rabbit Alexa Fluor 488 (Invitrogen), anti-mouse Alexa Fluor 594, and Alexa Fluor™ 594 Phalloidin at 1:40 dilutions (Invitrogen). Samples were then washed with PBS three times for 5 min each. Samples were then counter-stained with DAPI in Fluoroshield mounting medium (Sigma, St. Louis, MO, USA). The samples were analyzed by fluorescence microscopy (AXIO Imager D2, Zeiss) at a 20× magnification. A total depth of 20 µm was acquired for each sample and the total projection was visualized in the xy planes.
7
Bioactive PDMS Substrate for Fibroblast Culture
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The PDMS precursor was poured into a Petri dish and cured in an oven at 50°C for 12 h. The cured PDMS substrate was cut into 2.5 cm × 2.5 cm; then, half of the PDMS substrate was immersed in a solution of dopamine hydrochloride (0.5 wt%) and Tris–HCl (1 M, 10 mL, pH 8.5) for 3 days. After sterilization with ethanol (70%) and washing with water, the PDMS-based substrate was placed in a Petri dish containing culture media. Mouse NIH-3T3 embryonic fibroblasts (105 cells mL-1, ATCC) were seeded on the PDMS-based substrate and cultured in an incubator. After 3 days, the PDMS-based substrate was gently washed three times with water and was then stained with DAPI and rhodamine phalloidin for visualization. The fibroblasts on the PDMS-based substrate were imaged using fluorescence microscopy (FM; Axio Imager D2; ZEISS, Germany). After fixation with glutaraldehyde and freeze drying, the cells were imaged with scanning electron microscopy (SEM; S-4800; Hitachi, Tokyo, Japan).
8
Immunofluorescence Analysis of Phosphorylated Signaling Proteins
9
Confocal Imaging and Epifluorescence Protocols
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Confocal imaging was done on a Zeiss LSM 710. A 20 × /0.8 NA air objective was used. Images were acquired at 1 airy unit resolution with 4× undersampling of pixels in X–Y and 2× undersampling in Z. Excitation lasers were 488 nm (for Alexa Fluor 488 and fluorescein isothiocyanate (FITC)), 561 nm (for Alexa Fluor 594), and 633 nm (for Alexa Fluor 647). Emissions were collected as separate tracks over wavelength bands exclusive with other mutually excited fluorophores. Epifluorescence images were collected on an Axio Imager D2 (Carl Zeiss) with a 5× Fluor, 0.25 NA or 20× Plan-Apochromat, 0.8 NA objective, using a digital camera (Axiocam HRm, Carl Zeiss). AxioVision software was used to acquire images; Fiji (ImageJ) and Inkscape were used for image processing and analysis.
10
Chitosan Effect on Membrane Integrity
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An aliquot from each replica was taken, joined, and centrifuged at 5000× g for 10 min to evaluate chitosan’s effect on membrane integrity, immediately after 24 h of exposure. The pellet was stained with Sytox® Green (Thermo Fisher Scientific, Waltham, MA, USA) at a final concentration of 1nM for 30 min in the dark. The samples were observed under a fluorescence microscope (ZEISS, Axioimager D2, Jena, Germany) using the filter long pass for Fluorescein (450–490 for excitation and 515 nm for emission). Sytox® Green binds to nucleic acid, but it cannot penetrate the cell membrane. However, a damaged membrane allows the stain to infiltrate, resulting in a green fluorescence colour when analysed in a fluorescence microscope.
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