After CO2 asphyxiation, eyes were enucleated and placed in HBSS with Ca2+ and Mg2+ for dissection of the anterior chamber and removal of the RPE/sclera eyecup. After isolating the retina, four radial cuts were made in order for the retina to lie flat and the retina was placed outer segment side facing up in a 48-well plate containing FluoroBrite DMEM with anti-C1q Ab (1:100, ThermoFisher) labeled with quantum dots (SiteClick Molecular Probes) and incubated at 37 °C for 30 minutes. Ab solution was gently removed and retina was washed for 1 minute each in FluoroBrite DMEM. Finally, the well was filled with HBSS plus Mg2+ and Ca2+ with annexin V conjugated with Alexa Fluor 594 (1:1000, ThermoFisher) for 20 minutes before mounting the retina outer segment side up in FluoroBrite DMEM on a microscope slide with a glass coverslip. Slides were imaged with a Leica SPE upright confocal microscope.
Spe upright confocal microscope
Manufactured by Leica
The SPE upright confocal microscope is a high-performance laboratory instrument designed for advanced imaging applications. It offers a range of features and capabilities to capture detailed, high-quality images. The core function of the SPE upright confocal microscope is to provide optical sectioning and improved image quality through the use of a pinhole aperture, allowing for the examination of thick specimens with enhanced contrast and resolution.
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Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Dmrhc research microscope, by Leica (1 mentions)
Rat anti pecam1, by BD (1 mentions)
Donkey anti rat cy5, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Sp 1120, by Vector Laboratories (1 mentions)
5 protocols using spe upright confocal microscope
1
Retinal Immunostaining and Imaging
2
Immunostaining of Wing Disc in Drosophila
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The following antibodies were used: anti-β-Gal (mouse monoclonal antibody, 1/200, 40-1a, DSHB) and anti-Diap1 (mouse monoclonal antibody, 1/200, generous gift from B Hay). Third instar larvae were dissected in PBS pH 7.6, fixed in PBS-3.7% formaldehyde, washed three times for 10 min each in PBT (PBS, 0,3% Triton) and incubated with primary antibody overnight at 4 °C in PBT-FCS (PBS, 0,3% Triton, 10% FCS). Incubation with anti-mouse secondary antibody (Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) was carried in PBT-FCS for 2 h at room temperature. Larvae were then washed thrice in PBT. Finally, wing discs were mounted in CitifluorTM (Biovalley) and observed with a Leica SPE upright confocal microscope.
3
Apoptosis Detection in Drosophila Wing
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C96-Gal4, vg-Gal4 and ptc-Gal4 females were crossed with w1118, UAS-RBF, UAS-RBFD253A or UAS-bsk-RNAi males for apoptosis detection. Wing imaginal discs of the progeny were dissected in PBS pH 7.6, fixed in PBS/formaldehyde 3.7%, washed three times for 20 min in PBT (1X PBS, 0.5% Triton). Discs were then dissected, TUNEL staining was performed following manufacturer's instructions (ApopTag Red in situ apoptosis detection kit, Chemicon), and discs were mounted in CitifluorTM (Biovalley) and observed with a conventional Leica DMRHC research microscope using the N2.1 filter. For quantification experiments, discs were observed with a Leica SPE upright confocal microscope. White patches in the wing pouch were counted for at least 30 wing imaginal discs per genotype. Student's tests were performed and results were considered to be significant when α<5%.
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C96-Gal4, vg-Gal4 and ptc-Gal4 females were crossed with w1118, UAS-RBF, UAS-RBFD253A or UAS-bsk-RNAi males for apoptosis detection. Wing imaginal discs of the progeny were dissected in PBS pH 7.6, fixed in PBS/formaldehyde 3.7%, washed three times for 20 min in PBT (1X PBS, 0.5% Triton). Discs were then dissected, TUNEL staining was performed following manufacturer's instructions (ApopTag Red in situ apoptosis detection kit, Chemicon), and discs were mounted in CitifluorTM (Biovalley) and observed with a conventional Leica DMRHC research microscope using the N2.1 filter. For quantification experiments, discs were observed with a Leica SPE upright confocal microscope. White patches in the wing pouch were counted for at least 30 wing imaginal discs per genotype. Student's tests were performed and results were considered to be significant when α<5%.
4
Immunostaining and Imaging Yolk Sacs and BMDMs
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Yolk sacs were stained as described in Newton et al. [33 (link)]. Fixed tissues were stained with rat anti-PECAM-1 (BD, 550274) and rabbit anti-cleaved caspase-3 (CST, 9661). The following secondary antibodies were used: donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152) and donkey anti-rat Cy5 (Jackson ImmunoResearch, 712-175-153). Processed yolk sacs were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, P36392) and images were acquired using a LEICA SPE upright confocal microscope with 20x objective. On average about 50 optical sections were collected, representing about 50 μm deep volume, with 1.19 μm step size. The images shown are maximum intensity projections.
BMDMs were seeded at d5 on 4 chamber tissue culture treated glass slides (Falcon, 354104) and treated the following day. The cells were fixed in 4% PFA for 30 min at room temperature. Permeabilized in 0.25% Triton X-100 (w/v) and blocked in 5% BSA (w/v) and 0.05% Triton X-100 (w/v). Staining was performed with p65 (CST, 8242) for 2 h at room temperature followed by labeling with donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152) and 1:2000 Hoechst (H3569). The cells were mounted with ProLong Glass Antifade Mountant (P36980). Images were acquired using a LEICA SP8 inverted microscope with a 40x objective.
BMDMs were seeded at d5 on 4 chamber tissue culture treated glass slides (Falcon, 354104) and treated the following day. The cells were fixed in 4% PFA for 30 min at room temperature. Permeabilized in 0.25% Triton X-100 (w/v) and blocked in 5% BSA (w/v) and 0.05% Triton X-100 (w/v). Staining was performed with p65 (CST, 8242) for 2 h at room temperature followed by labeling with donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152) and 1:2000 Hoechst (H3569). The cells were mounted with ProLong Glass Antifade Mountant (P36980). Images were acquired using a LEICA SP8 inverted microscope with a 40x objective.
5
Chondrogenic Connexin Gap Junction Assay
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A tracer assay was performed to analyze the functionality of GJIC networks. Three replicates of each condition were seeded in chondrogenesis-inducing medium, as described above. After 6 days of differentiation, samples were washed with HBSS buffer without calcium or magnesium (Life Technologies, 14175095) and treated with neurobiotin 2% m v -1 (Vector, SP-1120) in HBSS for 90 s at 37˚C. Samples were then washed with HBSS, fixed with Formalin Solution, permeabilized with saponin and stained with Streptavidin-Texas Red conjugate (Life Technologies, S872) and Hoechst 1 µg mL -1 in BSA 1% m v -1 in PBS for 1 h at room temperature. Samples were imaged with a Leica SPE Upright Confocal Microscope as described above.
Images were analyzed with ImageJ software. A z-projection of each condensate was created, and background was removed. Distance of neurobiotin spread was measured in a straight line from the outer rim of the condensates' inwards, in at least two separate locations for each condensate.
Images were analyzed with ImageJ software. A z-projection of each condensate was created, and background was removed. Distance of neurobiotin spread was measured in a straight line from the outer rim of the condensates' inwards, in at least two separate locations for each condensate.
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