Proximity Ligation Assay was performed according to the manufacturer’s protocol (Duolink®, Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.25% Triton X-100. Samples were blocked with Duolink® Blocking Solution for 1 h at 37 °C in a humidity chamber. After removal of the blocking solution, primary antibodies diluted in Duolink® Antibody Diluent were added on the coverslips for 2 h at room temperature in a humidity chamber. Coverslips were washed 2× with Washing Buffer A. PLA plus and minus probes were put on in a 1:5 dilution in Duolink® Antibody Diluent for 1 h at 37 °C in a humidity chamber. Two washes with Washing Buffer A were followed by Ligase treatment in 1× Ligation Buffer for 30 min at 37 °C in a humidity chamber. Ligation buffer was tapped off and coverslips were washed twice with Washing Buffer A. Amplification was achieved by adding the Polymerase in 1× Amplification buffer for 100 min at 37 °C in a humidity chamber. After washing the samples 2× with 1× Washing Buffer B and 1× with 0.01× Washing Buffer B, coverslips were stained with 1 µg/ml Hoechst33342 and mounted using Dako mounting medium. Images were taken with a Leica SPE microscope using a ×63 1.4NA oil objective. The number of PLA spots per nucleus was quantified using Fiji/ImageJ (v1.51)89 .
Spe microscope
Manufactured by Leica
Sourced in Germany
The SPE microscope is a high-performance optical microscope designed for laboratory applications. It features advanced optical and mechanical components to provide clear, detailed images of samples. The SPE microscope is capable of various magnification levels and can be used for a range of scientific and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Vt1000, by Leica (2 mentions)
Spe confocal microscope, by Leica (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Axioskop upright microscope, by Zeiss (1 mentions)
Methyl salicylate, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Lumar v12 stereoscope, by Zeiss (1 mentions)
Moma 2, by Bio-Rad (1 mentions)
Oil red o, by Merck Group (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Insulin, by Merck Group (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Trolox, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Bafilomycin a, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by MP Biomedicals (1 mentions)
Aperio scanscope at2, by Leica (1 mentions)
Dmire2 inverted microscope, by Leica (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Eclipse 90i, by Olympus (1 mentions)
26 protocols using spe microscope
1
Proximity Ligation Assay for Protein Interactions
2
Microscopy Techniques in Biomedical Research
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Phase contrast microscopy was performed using Leica, Zeiss, and EVOS (Life Technologies) inverted fluorescence microscopes. Image analysis and manipulation was performed with ImageJ and GIMP. Histological sections were imaged on a Zeiss Axioskop upright microscope. Confocal microscopy was performed using a Leica SPE microscope.
3
Confocal Imaging of GFP-Labeled Cell Migration
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GFP-labeled MDA-MB-231 cells (Cell Biolabs Inc.) are maintained according to the vendor’s protocol. After embedding the cells, DIGME devices are kept in tissue culture incubator except when taken out for imaging. For confocal imaging, we use a Leica SPE microscope. 10X oil immersion objective is used when confocal reflection imaging of collagen fiber is needed. Otherwise, 4X air objective is used to image the fluorescently labeled cells and fluorescent particles embedded in the collagen matrix. The z-stacks of confocal imaging are taken with 2 μm z-steps. Confocal images are further processed in NIH ImageJ and MATLAB.
4
Immunofluorescence Imaging and In Situ Hybridization
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Cryostat sections were mounted in Vectashield (Vector Laboratories) or in 5 mg/mL propyl gallate in glycerol/PBS (9:1) with 0.01% azide. Whole mount explants were gradually dehydrated in methanol and cleared in methyl salicylate (Sigma) as described previously [31 (link),37 (link)]. Immunofluorescence images were taken on a confocal Leica SPE microscope, following imaging acquisition steps described previously [37 (link)]. Image analysis was performed using Fiji v. 1.49 (https://imagej.net/Fiji , accessed on 15 June 2022) software. Image histogram corrections and, when appropriate, maximum intensity projections of immunofluorescence confocal stacks were produced in Fiji. When applicable, contiguous images were stitched together into a single image using the pairwise stitching Fiji plugin [61 (link)]. Image acquisition of embryos and explants processed for in situ hybridization was performed using a Zeiss LUMAR V12 Stereoscope coupled to a Zeiss Axiocam 503 colour 3MP camera. Imaging of explants prior to in situ hybridization was performed in 50% formamide solution. For comparing the in situ hybridization patterns along the paraxial mesoderm, the Fiji plugin Straighten [62 (link)] was used and contralateral explant pairs were aligned by SIV.
5
Atherosclerotic Plaque Quantification in Murine Hearts
6
Visualizing E. histolytica Trophozoites and Cysts
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Propidium iodide staining. E. histolytica trophozoites or sarkosyl-treated cysts from a 52 mL culture were permeabilized in 1 mL 70% ethanol at 4 °C for 30 min with periodic vortexing, washed twice with PBS, resuspended in 1 mL PBS containing 0.2 mg/mL RNAse A, and stained with propidium iodide (50 µg/mL). Cells were stained for 15–20 min, rinsed once with PBS, and visualized microscopically.
WGA-488 staining. E. histolytica trophozoites or sarkosyl-treated cysts were stained with 1 mL of PBS containing 10 μg/mL AlexaFluor WGA-488 (1 mg/mL) for 20 min at ambient temperature with constant rotation. The cells were then rinsed with PBS and fixed in 1 mL 4% (v:v) paraformaldehyde for 15 min before imaging.
Imaging. Stained cells were imaged using a Leica SPE microscope. Cells stained with WGA-488 and propidium iodide were excited at 488 and 532 nm, respectively. Differential interference contrast (DIC) images were also taken.
WGA-488 staining. E. histolytica trophozoites or sarkosyl-treated cysts were stained with 1 mL of PBS containing 10 μg/mL AlexaFluor WGA-488 (1 mg/mL) for 20 min at ambient temperature with constant rotation. The cells were then rinsed with PBS and fixed in 1 mL 4% (v:v) paraformaldehyde for 15 min before imaging.
Imaging. Stained cells were imaged using a Leica SPE microscope. Cells stained with WGA-488 and propidium iodide were excited at 488 and 532 nm, respectively. Differential interference contrast (DIC) images were also taken.
7
Modulating Autophagy via RPS19 Silencing
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siRNAs against RPS19 were purchased from Invitrogen (#4392420). “Stealth RNAi” scrambled siRNAs from Invitrogen were used as a control (#12935-400). GFP-LC3 HEK cells were plated in 24-well plates (or on glass-bottom confocal dishes, Greiner Bio-One GmbH, #627870) the day before transfection with DMEM media. Transfections were done with Oligofectamine (Invitrogen, #12252-011) according to the manufacturer's instructions using the maximum recommended amount of siRNAs. Bafilomycin A (Sigma, #B1793) was added at a final concentration of 50 nM for 4 hours. Rapamycin (Sigma, #R8781) was added at a final concentration of 100 nM for 6 hours. 10 mM of Trolox (Sigma #238813) was added overnight. Insulin (Sigma, #I6634) was added at a final concentration of 350 nM for 6 hours. Confocal analysis was performed on a Leica SPE microscope. At least 8 shots per transfection were taken, and at least 3 transfections per condition were performed. The counter in ImageJ was used to quantify the number of puncta per cell and the number of cells with cytoplasmic GFP (the images were unlabeled to prevent bias).
8
Multimodal Imaging of Zebrafish Embryos
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Confocal data of whole mount immunohistochemical stainings a Leica (Germany) SPE microscope was used. Samples were mounted in glass bottom dishes (MaTek, Ashland, MA). Olympus (Germany) stereomircoscope was used for recording brightfield images of rx2::BMP4 hatchlings and the overview of the expression of rx2::GFPcaax. For whole mount in situ data acquisition, a Zeiss (Germany) microscope was used. Time-lapse imaging was performed with a Leica SP5 setup which was upgraded to a multi photon microscope (Mai Tai laser, Spectra Physics, Germany). It was recorded in single photon modus and multi photon modus. For time-lapse imaging, embryos were embedded in 1% low melting agarose and covered with zebrafish medium, including tricaine for anesthesia. Left and right eyes were used and oriented to fit the standard dorsal view or view from the side.
9
Perfusion and Tissue Preparation for Imaging
10
Multimodal Imaging of Neurological Markers
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Images of GFAP and D2R immunofluorescence were captured with a confocal Leica SPE microscope. For the mouse tissue, five z-stack images (total thickness 15 μm) were taken per region of interest (CA1, CA3 and DG region of the hippocampus and the striatum) at 40x magnification. For the human post-mortem tissue, 10 z-stack images (total thickness 5 μm) were acquired at 40x magnification in the CA1 region of the hippocampus. Post-mortem tissue sections which were stained for GFAP using immunohistochemistry were sent to the Histopathology/HIS facility at the Cancer Research UK Cambridge Institute and were imaged using an Aperio Scanscope AT2 (Leica Biosystems) at a 20x magnification, with a resolution of 0.503 µm per pixel.
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