Staining was performed on free-floating brain sections as previously described [37 (link)]. Sections were blocked with 0.2% (v/v) Triton X-100 and 5% (v/v) fetal bovine serum in PBS, and incubated overnight with mouse anti-PV (Millipore, Billerica, MA, MAB 1572; 1:1000) for PV staining or 6E10 (Signet, Dedham, MA; 1:800) for Aβ staining. PV and Aβ staining were visualized using anti-mouse Alex568-labeled secondary antibodies (Invitrogen, Carlsbad, CA; 1:400), incubated for 2 h at RT. Sections were washed and coverslipped in Vectashield including DAPI as a nuclear dye (Vector Laboratories, Burlingame, CA). PV staining was quantified using ImageJ v1.48. PV- and mCherrry-positive cells were counted using image thresholding and automated particle analysis in ImageJ (v1.48). All images were acquired on a Leica DM5000 fluorescence microscope using three different filter cubes (L5, TX2, and Y5) and with a DFC360FX camera (12 bits resolution). For each experiment, identical objective, exposure time, gain settings, and camera settings were used for all images.
Dm5000 fluorescence microscope
Manufactured by Leica camera
Sourced in United States
The Leica DM5000 is a fluorescence microscope designed for advanced imaging and analysis. It features a high-performance optical system, multiple illumination options, and a range of accessories to support various imaging techniques. The core function of the DM5000 is to enable detailed observation and capture of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Dfc360 fx camera, by Leica camera (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (1 mentions)
Ab7961, by Abcam (1 mentions)
C2562, by Merck Group (1 mentions)
Vectashield medium with dapi, by Vector Laboratories (1 mentions)
Sc 13024, by Merck Group (1 mentions)
Biotinylated goat anti rabbit, by Vector Laboratories (1 mentions)
P8825, by Abcam (1 mentions)
Anti cytokeratin, by Merck Group (1 mentions)
Avidin fitc, by Vector Laboratories (1 mentions)
Horse anti mouse texas red conjugated antibody, by Vector Laboratories (1 mentions)
Glass slide, by Thermo Fisher Scientific (1 mentions)
Iba 1, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
5 protocols using dm5000 fluorescence microscope
1
Immunohistochemical Staining for Parvalbumin and Amyloid-Beta
2
Bimolecular Fluorescence Complementation in Nicotiana
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Full-length ORFs with no stop codon of each test candidate (FAMA, FAMALGK, bHLH93, RBR, CYCD and CYCDLGK) were cloned into BiFC vectors (Walter et al., 2004 (link)) to generate fusion proteins with either N or C terminal half of the yellow fluorescence protein (YFP) fused to the C-terminus of the test candidate. FAMA and bHLH93 constructs were reported in (Ohashi-Ito and Bergmann, 2006 (link)). Assays were performed in Nicotiana benthamiana leaves as described in Ohashi-Ito and Bergmann (2006) (link). BiFC signals were visualized on a Leica DM5000 fluorescence microscope and quantified as percentage of YFP-positive nuclei over total number of pavement cells in a field of view (centered on the injection site). Results from three experiments are presented in Figure 1G .
3
Cell Viability Assay with PSPD3R Treatment
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Cell viability was determined by EdU (RiboBio, Guangzhou, China) assay.23 (link) The cells were plated at 1 × 104 cells per well in 96-well microtiter plates with 100 μL complete culture medium and treated with designated PSPD3R concentrations (0, 25, 50, and 75 μM) for 24 h at 37°C in a humidified chamber. Treatment with each PSPD3R concentration was replicated in three wells. After incubation, the cells were fixed with 4% paraformaldehyde for 30 min and then exposed to Apollo staining reaction solution. Nuclei were stained with Hoechst33342 Staining Solution, and images were captured using a DM5000 fluorescence microscope (Leica, Buffalo Grove, IL, USA).
4
Immunohistochemical Analysis of Kidney Sections
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Five micron-thick kidney sections were de-paraffinized with xylene and hydrated with graded ethanols. The antigen unmasking and labeling was performed as previously described (Sharma et al., 2005 (link)). Briefly, kidney sections were incubated with 1 M ammonium chloride for 30 min to quench autofluorescence, followed by washing in PBST, and then blocked in 10% normal goat serum (NGS) or 10% normal horse serum (NHS) for 1 h at room temperature. Sections were treated with rabbit anti-Cux1 (1:50, Santa Cruz, sc-13024), mouse anti-PCNA (1:3000, Sigma, P8825), or rabbit anti-p27 (1:100, AbCam, ab7961) primary antibodies overnight at 4 °C. Biotinylated goat anti-rabbit (1:400, Vector) secondary antibody was used to detect Cux1 and p27. The sections were then subsequently probed with FITC-avidin (5 μg/ml, Vector). PCNA antibodies were detected using a horse anti-mouse Texas Red conjugated antibody (Vector, 1:400). To identify collecting ducts, kidney sections were labeled with anti-cytokeratin (1:400, Sigma, C2562). Sections were washed in PBST after the antibody treatments, mounted with Vectashield medium with DAPI (Vector) and slides were viewed on a Leica DM5000 fluorescence microscope and images captured with a Leica DFC365 digital camera.
5
Immunostaining of Lumbar Spinal Cord
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The lumbar spinal cord was transected into 10-μm-thick sections using a cryostat and 8–10 slices were mounted directly onto a glass slide (Fisher Scientific). Sections were washed on the glass slides in 0.01 M paraformaldehyde/PBS and then blocked with 3% donkey serum for 1 h. Sections were incubated overnight with a mouse antibody against HDAC2 (1:100, Abcam), and rabbit antibodies against Iba-1 (1:500, Abcam), NeuN (1:300, Abcam), or GFAP (1:600, Abcam). The sections were then washed three times with PBS, then incubated in the dark for 2 h with a donkey anti-mouse red fluorescent-antibody (1:200, Jackson) or donkey anti-rabbit green fluorescent-antibody (1:200, Jackson). PBS was used instead of the primary antibody as a negative control. Immunofluorescence was visualized and digitally captured using a Leica DM5000 fluorescence microscope.
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