Human AD or age-matched control brains were incubated in 10% neutral buffered formalin for 48 h and then dehydrated and embedded in paraffin. Prior to immunostaining, slides were deparaffinized by oven heating and immersion in xylene. After dehydration through graded alcohols and water, tissue slices were immunostained overnight with a primary antibody against Nrp-2 (Cell Signaling Technology, MA, USA) at 1:50, followed by Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, CA, USA) at 1:100 After three washes in permeabilization buffer and a wash in PBS, cells were mounted on microscope slides in mounting medium (DAKO, CA, USA). Confocal microscopy was performed using an LSM 510 (Carl Zeiss, Jena, Germany).
Microscope slide
Manufactured by Agilent Technologies
Sourced in United States
Microscope slides are transparent plates made of glass or plastic used to hold samples for examination under a microscope. They provide a flat, stable surface to mount and view specimens.
Automatically generated - may contain errors
Lab products found in correlation
Mounting medium, by Agilent Technologies (5 mentions)
Lsm 510, by Zeiss (2 mentions)
Laser scanning microscope 5 pascal, by Zeiss (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Cryostar nx70, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by ITW Reagents (1 mentions)
12 mm circle cover slips, by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting media, by Agilent Technologies (1 mentions)
Triton 100x, by Merck Group (1 mentions)
Tsc spe confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Laser scanning microscope 5 pascal program, by Zeiss (1 mentions)
7 protocols using microscope slide
1
Immunostaining of Neuropilin-2 in Human Brains
2
Mitochondrial Ca2+ Imaging in Cells
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Mitochondrial Ca2+ levels were monitored using Rhod-2 AM (Mészáros et al., 2012 (link)). Cells were treated with shikonin for 48 h; harvested, washed, resuspended in PBS containing 1 µM Rhod-2 AM; and incubated for 15 min at 37°C. Subsequently, the cells were washed and suspended in PBS for further analysis by flow cytometry. To confirm the flow cytometry results, the cells were seeded in 4-well chambers, and image analysis was conducted by loading cells with Rhod-2 AM for 30 min at 37°C. After washing, the stained cells were mounted on microscope slides with mounting medium (DAKO, Carpinteria, CA, USA). Images were captured on a confocal microscope using the Laser Scanning Microscope 5 PASCAL software (Carl Zeiss, Jena, Germany).
3
Quantifying Amyloid Plaque Deposition
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Each mouse was anesthetized in an isoflurane chamber with a 5% isoflurane concentration at a flow rate of 2 L/min and the brain was decapitated using a guillotine. The brain was quickly frozen in dry ice and stored at −80 °C until further use for biochemical analyses. One hemisphere of the brain was cut in the sagittal plane into 30 µm thick slices using a cryotome (CryoStar NX70, Thermofisher). These 30 µm slices were incubated with methoxy-X04 solution (0.004 mg/ml, Tocris Bioscience, 1:1 ethanol-PBS solution) at room temperature for 30 min. The unspecific dye was removed by washing it three times with a 1:1 Ethanol-PBS solution and two times with Milli-Q water, and the slices were then mounted on microscope slides with a fluorescent mounting medium (Dako, Germany). 3D image stacks were obtained on an epi-fluorescence microscope (Axio Imager.M2 with ApoTome.2, Jena, Zeiss, Germany). Tile scan mode was used to image the brain slices which also allows the automatic stitching of the different brain segments. The excitation wavelength for the methoxy staining was fixed at 405 nm, and the emitted light was collected from 410 to 585 nm. The area and number of plaques were counted after the removal of the background using Fiji (ImageJ).
4
Dendrimer Internalization in Cells
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Internalization of the dendrimers into cells was analyzed by confocal microscopy with a Leica TSC SPE Confocal Microscope (Leica, Wetzalar, Germany). PBMCs were seeded at a density of 1 × 106 cells/well in 24-well plates and U87MG-CD4+CCR5+ cells were seeded at 7.5 × 103 cells in 12 mm circle cover slips (Thermo Fisher Scientific, Waltham, MA, USA) pre-treated for 24 h with poly-L-Lysine (Sigma, St Louis, MO, USA). Cells were treated with the fluorescent dendrimers for 1 h, 2 h or 6 h at 37ºC. After incubation, handling PBMCs as suspension cells and U87MG-CD4+CCR5+ as adherent cells, cells were rinsed twice with 3% bovine serum albumin (BSA, Sigma, St Louis, MO, USA) phosphate buffered saline (PBS, Lonza, Base, Switzerland), fixed with 4% paraformaldehyde (PFA, Panreac, Barcelona, Spain) and permeabilized with 0.1% Triton 100X (Sigma, St Louis, MO, USA) for 15 min. Cells were then incubated with Alexa Fluor® 555 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT for actin labelling and then rinsed with PBS 3% BSA. Lastly, cells were incubated with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, St Louis, MO, USA) for nuclear visualization and mounted in microscope slides (Dako, Carpinteria, CA, USA) with fluorescent mounting media (Dako, Carpinteria, CA, USA).
5
Intracellular ROS Detection by DCF-DA
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Cells were seeded in six-well plates at a density of 3 × 105 cells/well. Cells were treated with 25 μM dichlorodihydrofluorescein diacetate (DCF-DA) and the fluorescence of 2′,7′-dichlorofluorescein (DCF) was detected using a flow cytometer (Becton Dickinson) and analyzed using CellQuest software. Image analysis for the generation of intracellular ROS was achieved by seeding cells on a coverslip-loaded six-well plate at a density of 2 × 105 cells/well. Then, 100 μM DCF-DA was added to each well and cells were incubated for an additional 30 min at 37°C. After washing with PBS, stained cells were mounted onto a microscope slide in mounting medium (DAKO). Images were acquired using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss) on a confocal microscope.
6
Calcium Signaling in UVB-Induced Stress
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Cells were seeded in a 4-well chamber slid at a density of 1 × 105 cells/mL. At 16 h after plating, cells were treated with 30 μM BDB, and then exposed to UVB 1 h later. After an additional incubation for 24 h, the cells were loaded with 10 μM Fluo-4-AM (Molecular Probes, Eugene, OR, USA), a Ca2+-sensitive fluorescent probe, and incubated for 30 min at 37 °C. This was followed by washing with phosphate-buffered saline to remove any unbound dye. The stained cells were mounted onto a microscope slide with mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were collected using the FV1200 laser scanning microscopes Olympus FV10-ASW viewer 4.2 (Olympus Corporation, Tokyo, Japan) on a confocal microscope. For flow cytometry, cells were seeded in 6-well plates at a density of 0.8 × 105 cells/well. The cells were then treated under the same conditions. The cells were suspended in PBS containing Fluo-4-AM (10 μM). After incubation at 37 °C for 30 min, analysis was performed. The detection of Ca2+ level was repeated three times.
7
Evaluation of Intracellular ROS Generation
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Cells were seeded in 6 well plates at a density of 3 × 105 cells/ml. After 24 h of incubation at 37°C, the cells were treated for 24 h with 140 μM 5-FU. Cells were treated with 20 μM dihydrorhodamine (DHR) 123, and the DHR123 fluorescence was detected using a flow cytometer (Becton Dickinson, Mountain View, CA, USA) and analyzed using Cell Quest software. For image analysis of the generation of intracellular ROS, cells were seeded on a coverslip-loaded 6-well plate at a density of 2 × 105 cells/ml. After addition of 20 μM DHR123 to each well and incubation for an additional 30 min at 37°C, plates were washed with PBS, and stained cells were mounted onto a microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images were analyzed using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope.
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