For retinal whole mounts, excess tissues were dissected along with the cornea and lens. Eye cups were blocked in 5% normal goat serum in 0.25% Triton X-100 for overnight at 4°C. Eye cups were incubated with an anti-GFP antibody (1:100, Abcam) for 48 hours. Subsequently, eye cups were washed with PBS in 0.25% Triton X-100 and incubated in anti-mouse Alexa Fluor 555 (1:200, Abcam) for 24 hours at 4°C. After adequate washing, 4 radials cuts were made along with the optic disc and retina was dissected and mounted with FluorSave™ (Sigma-Aldrich). Whole mounts were imaged in a Lecia confocal microscope (DMi8, Wetzlar, Germany).
Dmi8 confocal microscope
Manufactured by Leica camera
Sourced in Germany, China
The DMi8 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular design, allowing for the integration of various optical and detection components to suit specific research needs. The DMi8 utilizes confocal technology to provide optical sectioning, enabling the capture of high-resolution, three-dimensional images with improved contrast and clarity compared to traditional wide-field microscopy.
Automatically generated - may contain errors
Lab products found in correlation
X applications suite, by Leica camera (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Polyfreeze, by Merck Group (4 mentions)
Donkey anti goat cy3 antibody, by Jackson ImmunoResearch (4 mentions)
Normal donkey serum (nds), by Santa Cruz Biotechnology (4 mentions)
Hcx plapo 40x na 1.25, by Leica camera (4 mentions)
Anti gfp antibody, by Abcam (2 mentions)
Fluorsave, by Merck Group (2 mentions)
Alexa fluor 555, by Abcam (2 mentions)
Las x software, by Leica camera (2 mentions)
Goat anti human fix antibody, by Affinity Biologicals (2 mentions)
Fluoroshield with dapi, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Donkey anti rabbit alexa fluor 594 a21207, by Thermo Fisher Scientific (2 mentions)
Anti iba1 019 19741, by Fujifilm (2 mentions)
Ca2 and mg2 ions, by Thermo Fisher Scientific (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Microtome, by Leica camera (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
99 protocols using dmi8 confocal microscope
1
Retinal Whole Mount Immunostaining
2
Retinal Whole Mount Immunostaining
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For retinal whole mounts, excess tissues were dissected along with the cornea and lens. Eye cups were blocked in 5% normal goat serum in 0.25% Triton X-100 for overnight at 4°C. Eye cups were incubated with an anti-GFP antibody (1:100, Abcam) for 48 hours. Subsequently, eye cups were washed with PBS in 0.25% Triton X-100 and incubated in anti-mouse Alexa Fluor 555 (1:200, Abcam) for 24 hours at 4°C. After adequate washing, 4 radials cuts were made along with the optic disc and retina was dissected and mounted with FluorSave™ (Sigma-Aldrich). Whole mounts were imaged in a Lecia confocal microscope (DMi8, Wetzlar, Germany).
3
Immunofluorescent Staining of Coronavirus Spike Proteins
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VERO, A549, and MDCK cells grown on coverslips were analyzed by immunofluorescent staining. Cells were fixed with 4% paraformaldehyde in PBS for 25 min at RT after which permeabilization was performed using 0,1% Triton in PBS. Subsequently, the coronavirus spike proteins were applied at 50μg/ml for 1 h at RT. Primary Strep-MAb classic chromeo-488 (IBA) and secondary Alexa-fluor 488 or 555 goat anti-mouse (Invitrogen) were applied sequentially with PBS washes in between. DAPI (Invitrogen) was used as nuclear staining. Samples were imaged on a Leica DMi8 confocal microscope equipped with a 10x HC PL Apo CS2 objective (NA. 0.40). Excitation was achieved with a Diode 405 or white light for excitation of Alexa488 and Alexa555, a pulsed white laser (80MHz) was used at 488 nm and 549 nm, and emissions were obtained in the range of 498-531nm and 594–627 nm respectively. Laser powers were 10–20% with a gain of a maximum of 200. LAS Application Suite X was used as well as ImageJ for the addition of the scale bars.
4
Confocal Imaging of Turn-on Probe HX103
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For confocal fluorescence imaging experiments, about 10000 cells in 200 µL of medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin were seeded in each well of an eight-well chamber (µ-Slide 8 well, Ibidi, #80826) and cultured for 24–48 h. The medium was replaced with 200 µL of serum-free medium (RPMI-1640 or DMEM) containing 5 µM fluorescent turn-on probe HX103 (0.1% DMSO). The cells were incubated with the probe at 37 °C for 1 h in a humidified incubator under 5% CO2 in the air, followed by washing with PBS (three times). The cells were also co-stained with nuclear dye (Hoechst 33342, 1 µg/mL, 20 min, Invitrogen). For competition experiments, gefitinib was in situ pre-incubated for 1 h prior and then co-incubated with HX103 for another 1 h. The differential interference contrast (DIC) and fluorescence images were obtained using a Leica DMi8 confocal microscope. A white light laser was used for the light source in the confocal microscope. The fluorescence of HX103 was excited with 440 nm with emission collected at 500–550 nm. The fluorescent intensity of the confocal images was analysed by Leica LAS X software.
5
Immunofluorescence Imaging of Pancreatic Cells
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MIN6 cells were plated in glass-bottom dishes and treated with PCs for the indicated times. The culture media was removed, and the cells were washed with cold PBS three times. Then, the cells were fixed in 4% paraformaldehyde for 30 min, followed by permeabilization of cells with 0.1% Triton X-100 for 15 min. Cells were stained with Hoechst 33342 (5 mg/mL) for 15 min, and the nuclei were observed under a Leica DMi8 confocal microscope.
6
Immunostaining of Human FIX in Murine Liver
7
Whole Gut Confocal Microscopy
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Microscopy was conducted with a Leica DMi8 confocal microscope using either a 40× (1.30 NA) HC Plan Apo or a 60× (1.40 NA) HC Plan Apo oil immersion objective. Laser lines were generated using a white-light laser with AOTF crystal, and excitation wavelengths for fluorophores were: mGFP5, 488 nm; mCherry, 591 nm; Cy5, 650 nm. Whole gut images were generated by tiling multiple captures then merging using the Mosaic Merge function in the Leica Application Suite LAS X to stitch into a single stack. Z-stacks for whole guts were 70–80 µm in thickness with slices every 0.5 µm or less. To render two-dimensional images for publication, fluorescence channels were processed as maximum intensity z-projections and the brightfield channel is represented by a single z-slice from the middle of the stack.
8
Phagocytic Activity of Aged Microglia
9
Plakoglobin Immunofluorescence in T47D Cells
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T47D cells (5×105) and T47D cell clusters grown in 24-well plates were fixed with 4 % paraformaldehyde for 30 min, then washed with PBS three times and stored at 4 °C. After 24 h, cells were permeabilized with 0.5 % TritonX-100 for 5 min, followed by washing with PBS. Cells were then incubated with an anti-human plakoglobin antibody (CST) at 4 °C overnight. The next day, cells were incubated with the secondary antibody (goat anti-rabbit IgG (H+L) conjugated to cy3 Fluor dye (Invitrogen)) for 30 min, and treated with PBS and a gradient of ethanol for 5 min each. The cells were incubated with DAPI for 5 min and covered with coverslips. Finally, they were visualized and recorded using a Leica DMi8 confocal microscope. After secondary antibody addition, all steps were performed in dark conditions.
10
Neuronal Quantification in Mouse Brain
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At t = 12 days post-injection, mice were deeply anaesthetized with Fatal-Plus at a dose of 90 mg kg−1 and transcardially perfused with 20 ml of PBS, followed by 20 ml of 4% paraformaldehyde solution. Brains were quickly extracted and stored in 4% paraformaldehyde solution at 4 °C for 24 h, and were then transferred to 30% sucrose in PBS solution and allowed to equilibrate for 2 days. Brains were then mounted on a cryostat using OCT and sectioned coronally (50 µm). The floating sections were washed in PBS and stained for neurons using anti-NeuN antibody (Sigma-Aldrich MAB377; 1:500) and Alexa 488 secondary antibody (ThermoFisher A11001; 1:1,000). The sections were mounted on slides with PVA-DABCO. Images were acquired using a Leica DMi8 confocal microscope with a 10× and 20× air objective.
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