Wild type and Dynlrb1+/- DRG ganglia were dissociated as previously described and cells were plated on coverslips in F12 medium (ThermoFisher Scientific). 48 h after plating cells were washed in PBS, fixed in 4% PFA in PBS for 30 min and immunostained for β−catenin (1:100 dilution), NFH (1:2,000 dilution) and DAPI as described previously. Coverslips were then mounted with Flouromount-GTM and imaged using a confocal laser-scanning microscope (Olympus FV1000, 60x oil-immersion objective Olympus UPLSAPO - NA 1.35). Image quantification was performed using ImageJ software. Nuclei were identified using a mask drawn on the DAPI straining and cytoplasm was masked using the NFH staining. Nuclear and cytoplasmic intensity of β−catenin was recorded. Statistical analysis was carried out with a one-way ANOVA followed by Tukey post-hoc multiple comparison test using the statistical software GraphPad Prism.
Uplsapo na 1
Manufactured by Olympus
Sourced in Japan
The UPLSAPO - NA 1.35 is a high-numerical aperture (NA) objective lens designed for use in optical microscopy. It is engineered to provide high-resolution imaging and excellent light-gathering capabilities. The core function of this objective lens is to efficiently collect and focus light from the sample under observation, enabling detailed visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000, by Olympus (4 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Uplanfln, by Olympus (1 mentions)
Life 888 emccd camera, by Oxford Instruments (1 mentions)
Ix73 fluorescence microscope, by Olympus (1 mentions)
Dp26 camera, by Olympus (1 mentions)
8 protocols using uplsapo na 1
1
Quantification of β-catenin in DRG neurons
2
Quantifying Adenoviral Cre-GFP Transduction in DRG Neurons
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The efficiency transduction of adenovirus 5 Cre-GFP (Ad5 Cre-GFP) and the efficiency of the viral Cre were tested by infecting DRG neurons with Ad5 Cre-GFP at a multiplicity of infection (MOI) of 100. DRG neurons from Ai9 mice (Madisen et al., 2010 (link)) were dissected as previously described. The Ai9 Cre reporter mice harbour a LoxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (td-Tomato). The virus was added directly into the F12 culture medium immediately after plating. Infected cultures were then incubated at 37°C for 48 and 72 h. After each time-point, neurons were washed in PBS, fixed in 4% PFA in PBS for 30 min and immunostained with an α-GFP antibody (1:1000) as previously described. Coverslips were then mounted with Flouromount-GTM and imaged using the ImageXpress (Molecular Devices) automated microscope at 10X magnification or using a confocal laser-scanning microscope (Olympus FV1000, 60x oil-immersion objective Olympus UPLSAPO - NA 1.35). Image quantification was performed using the proprietary software Metamorph (Molecular Devices). Statistical analysis was carried out with a one-way ANOVA followed by Tukey post-hoc multiple comparison test using the statistical software GraphPad Prism.
3
Neuromuscular Junction Staining in Mice
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Gastrocnemius muscles from wild type and RNX3-Dynlrb1-/- mice were sectioned at a thickness of 50 μm. Staining of the neuromuscular junction was achieved as previously described by incubation with Alexa 555-α-bungarotoxin (1:1000 dilution). Sections were then imaged using a confocal laser-scanning microscope (Olympus FV1000, 60x oil-immersion objective Olympus UPLSAPO - NA 1.35) and the number of positive NMJ-plate per area were manually counted and normalized by the area. Statistical analysis was performed with t-test using the statistical software GraphPad Prism.
4
Cryosectioning and Immunostaining of Neuronal Tissues
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Tissue samples from muscle, skin, DRGs and sciatic nerve were harvested and fixed in 4% PFA in PBS (30 min for skin at RT, overnight at 4°C for muscles, DRG ganglia and sciatic nerve). The tissues were then washed in PBS and equilibrated in 30% w/v sucrose in PBS, embedded in Optimal Cutting Temperature Compound (O.C.T., Tissue-Tek®) and finally cryo-sectioned at a thickness ranging from 10-50 μm. Sections were then re-hydrated in PBS, blocked and permeabilized with 5% goat serum, 1% BSA, 0.1% Triton X-100 in PBS, or donkey serum, 0.1% Triton X-100 in PBS for 1 h and incubated with primary antibody overnight at 4°C. Sections were then washed 3 times in PBS and incubated for 2 h with secondary antibodies from Jackson Immunoresearch (Alexa Fluor 647 or Alexa Fluor 488 conjugated, 1:500 dilution in PBS). Slides were then washed in PBS, mounted with Flouromount-GTM, and imaged using a confocal laser-scanning microscope (Olympus FV1000, 60x oil-immersion objective Olympus UPLSAPO - NA 1.35).
5
Immunohistochemical Analysis of Neuronal Markers
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SNs were fixed in 4% PFA overnight at 4°C. The next day, nerve segments were dehydrated by increasing concentrations of ethanol, embedded in paraffin, and 5-μm cross sections were taken. The slides then underwent deparaffinization with xylene and ethanol, followed by antigen retrieval in tris-EDTA buffer [10 mM tris base, 1 mM EDTA, and 0.05% Tween 20 (pH 9.0)] using a pressure cooker at 125°C for 1 min with subsequent cooling to RT for 1 hour. After two washes in PBS, blocking was performed in 20% horse serum and 0.2% Triton in PBS for 1 hour at RT. Primary Abs (TUJ1, PTBP1, and PTBP2) were incubated in AB solution (2% horse serum and 0.2% Triton in PBS) overnight at RT. Secondary Abs (donkey, 1:500, Jackson ImmunoResearch) were applied in AB solution for 2 hours at RT. Slides were mounted in Fluoromount (F4680, Sigma-Aldrich), and confocal images were taken using an Olympus FV1000 Confocal laser-scanning microscope at ×60 magnification with oil-immersion objective (Olympus UPLSAPO, NA 1.35).
6
Whole Tissue Staining Optimization Protocol
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Whole tissue staining was performed as described (59 (link)) with minor adjustments. Tissue was fixed in 4% PFA o/n at 4°C and washed in PBS. Subsequently, samples were dehydrated in methanol/H2O series (20, 40, 60, 80. and 100 for 1 hour each), bleached in fresh 5% H2O2 in methanol o/n at 4°C and rehydrated the next day. After two washes in PTx.2 buffer (0.2% Triton X-100 in PBS), samples were permeabilized in PTx.2 containing glycine (23 mg/ml) and 20% (v/v) dimethyl sulfoxide (DMSO) for 2 days at RT. Blocking was performed in PTx.2 containing 6% donkey serum and 10% DMSO for 3 days at RT. Abs for PTBP1, PTBP2, and NFH were applied in in PTwH buffer [0.2% Tween 20 and heparin (0.01 mg/ml) in PBS] supplemented with 5% DMSO and 3% donkey serum for 3 days at RT. After five washes (each 30 min) in PTwH buffer, tissue samples were incubated with secondary Abs in PTwH with 3% donkey serum for 2 days at RT, followed 1 hour DAPI in PTwH and five washes in PTwH (30 min). Last, samples were transferred to 75% glycerol for 1 week before imaging. For imaging, an Olympus FV1000 Confocal laser-scanning microscope at ×60 magnification with oil-immersion objective (Olympus UPLSAPO, NA 1.35) was used.
7
Live Cell Microscopy Imaging Protocol
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Pictures of living cells were acquired using an Olympus IX73 fluorescence microscope fitted with a 40 X objective (0.75NA UPlanFLN, Olympus) or a 20 × 0.40 NA objective (LCAChN 0.4NA, Olympus), a X-Cite Series 120Q lamp (Lumen dynamics), a DP26 camera (Olympus) and using the adequate filters set. For time series, cells were seeded in glass-bottom dishes (from MatTek or ibidi µSlide) in phenol red-free Leibovitz’s L-15 medium containing 10% FBS and pen./strep. For each time point, z-stacks were acquired using a Andor/Olympus Yokogawa CSU-X1 confocal spinning disk fitted with 60 X (UPLSAPO NA 1.35, Olympus) or 100 X (UPLSAPO NA 1.4, Olympus) objectives, a Andor iXon Life 888 EM-CCD camera and with temperature and humidity control. The white scale bars on representative pictures represent 10 µm.
8
Fluorescence Microscopy Analysis of Sperm
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The samples were observed under an Olympus FlowView confocal fluorescence microscope (Olympus FV1000, 60x oil-immersion objective Olympus UPLSAPO - NA 1.35, Tokyo, Japan). The lasers used for excitation were 488 nm (emission range 501–546 nm) and 560 nm (emission range 575–675 nm) with 10% transmissivity and 2 airy units of confocal pinhole. Images were visualized with FV10–ASW 4.2a software (Olympus, Tokyo) and analyzed by ImageJ (Win-64 version 7.1) with Z Projection (Max Intensity) and background subtraction using rolling ball (radius = 50 pixels). Regions of interest (acrosome, equatorial ring, postnuclear cap, midpiece and tail) were manually defined by differential interference contrast (DIC) images, and the mean fluorescence intensity per area unit was measured in each of them.
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