Cells were seeded onto glass coverslips, grown for 24 h then treated with doxycycline for another 24 h. Cells were rinsed twice with ice-cold PBS, fixed with methanol for 20 min at −20 °C, and washed four times with cold PBS. The cells were incubated with 10% goat serum in PBS for 20 min and were then incubated in primary antibodies overnight for BirA∗ (Novus Biologicals #6C4c7, 1:400 dilution) in 10% goat serum in PBS. After two PBS washes, the cells were incubated with AlexaFluor 568 goat anti-mouse (Invitrogen # A-11004, 1:800) at room temperature for 1 h. Following two more PBS washes, we stained the nuclei with 4',6-diamidino-2-phenylindole (1 μg/μl) for 10 min at room temperature, washed them twice with PBS and mounted them with Immuno Mount (Thermo Fisher Scientific). GFP-DDB1–expressing cells were fixed with 4% paraformaldehyde, and we stained the nuclei with 4',6-diamidino-2-phenylindole (1 μg/μl) for 10 min at room temperature, washed them twice with PBS, and mounted them with Immuno Mount (Thermo Fisher Scientific).
Immuno mount
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Immuno-mount is a water-soluble, non-fluorescent mounting medium designed for use with immunohistochemistry and immunofluorescence procedures. It is formulated to preserve the antigenicity and fluorescence of labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Eclipes 80 i, by Nikon (3 mentions)
Eclipse 80i, by Nikon (2 mentions)
Lysis buffer, by GenDEPOT (2 mentions)
Infinite m200 pro microplate reader, by Tecan (2 mentions)
Mouse anti dsred antibody, by Takara Bio (2 mentions)
Rabbit anti npy primary antibodies, by Merck Group (2 mentions)
Evos imaging system and microscope, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 goat anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Em fcs cryochamber, by Leica (2 mentions)
Ultracut uct ultramicrotome, by Leica (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
2 cm2 coverslips, by Merck Group (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Bx60 microscope, by Olympus (2 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
42 protocols using immuno mount
1
Immunocytochemical Analysis of BirA* and GFP-DDB1
2
Evaluating Photosensitizer Uptake in Cancer Cells
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Cancer cells (3 × 105 cells) seeded on the cover glass in six-well plates were exposed to Ce6 or nanophotosenstitizer for 90 min. After that, the cells were washed with PBS twice and then fixed with immobilization solution (Immunomount, thermo Electron Co. Pittsburgh, PA, USA). The cells were observed with fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan).
3
Fluorescent Imaging of DOX-Nanoparticle Uptake
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For fluorescence observation of cells, 3 × 105 HuCC-T1 cells were seeded in 6 wells with cover glass. These were treated with free DOX or DOX-incorporated ChitoHISss nanoparticles for 60 min. After that, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed again with PBS, and then immobilized with mounting solution (Immunomount, Thermo Electron Co., Pittsburgh, PA, USA). The cells were observed with a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan). Each measurement from fluorescence observations and analysis was repeated at least three times and then presented as an average image.
4
Photosensitizer Uptake in HeLa Cells
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HeLa cells (3 × 105) were seeded in 6-well plates with cover glass and then treated with either Ce6 or nanophotosenstitizers for 90 min. After that, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were washed with PBS twice and then immobilized with mounting solution (Immunomount, Thermo Electron Co., Pittsburgh, PA, USA). The cells were observed with a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan).
5
Cellular Uptake and Fluorescence Imaging of Photosensitizers
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The cancer cells (2 × 104 cells) were seeded in a 96-well plate and then exposed to Ce6 or nanophotosensitizers for 2 h as described above. The cells were washed with PBS twice and solubilized in 50 µL of lysis buffer (GenDEPOT, Barker, TX, USA). Then, the Ce6 uptake ratio was measured with an Infinite M200 pro microplate reader (Tecan) (excitation wavelength: 407 nm; emission wavelength: 664 nm).
For fluorescence observation, 3 × 105 cells seeded in 6 well plates with cover glass were exposed to Ce6 or nanophotosenstitizers for 90 min. The cells were washed with PBS twice, fixed with a 4% paraformaldehyde solution and then immobilized with a mounting solution (Immunomount, thermo Electron Co. Pittsburgh, PA, USA). Observation of the cells was performed with a fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan).
For fluorescence observation, 3 × 105 cells seeded in 6 well plates with cover glass were exposed to Ce6 or nanophotosenstitizers for 90 min. The cells were washed with PBS twice, fixed with a 4% paraformaldehyde solution and then immobilized with a mounting solution (Immunomount, thermo Electron Co. Pittsburgh, PA, USA). Observation of the cells was performed with a fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan).
6
Quantifying Ce6 Uptake in HeLa Cells
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2 × 104 HeLa cells in 96 wells were cultured overnight in 5% CO2 incubator at 37 °C. Ce6 or nanophotosensitizers were exposed to cells for 2 h as similarly described in PDT treatment. After washing of cells with PBS, cells were solubilized in 50 µL of lysis buffer (GenDEPOT, Barker, TX, USA) to measure Ce6 uptake ratio. Infinite M200 pro microplate reader (Tecan) (excitation wavelength: 407 nm, emission wavelength: 664 nm) was employed to measure Ce6 contents in cells.
For observation of cells using fluorescence microscope, HeLa cells (3 × 105) in six wells with cover glass were treated with Ce6 or nanophotosenstitizers. 60 min later, cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min. This was washed with PBS again to remove byproducts. Mounting solution (Immunomount, Thermo Electron Co., Pittsburgh, PA, USA) was used to immobilize these cells. After that, fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan) was used to observe cells.
For observation of cells using fluorescence microscope, HeLa cells (3 × 105) in six wells with cover glass were treated with Ce6 or nanophotosenstitizers. 60 min later, cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min. This was washed with PBS again to remove byproducts. Mounting solution (Immunomount, Thermo Electron Co., Pittsburgh, PA, USA) was used to immobilize these cells. After that, fluorescence microscope (Eclipes 80i; Nikon, Tokyo, Japan) was used to observe cells.
7
Islet Cell Localization in Wistar and GK Rats
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Localization of insulin and glucagon in pancreatic islets of control Wistar and GK rats was performed by a doublelabeled immunofluorescence method as described by Adeghate et al. [24] . Briefly, pancreatic tissue pieces were fixed overnight in Zamboni's solution, dehydrated in graded concentration of ethanol, cleared using xylene and embedded in paraffin wax at 55°C. Sections of 5 µm thickness were cut on a microtome (Shandon AS325, Pittsburg, PA, USA), deparaffinized with sequential changes in xylene, and rehydrated with descending concentrations of ethanol. This was followed by antigen retrieval, conducted by boiling the slides in citrate buffer followed by gradual cooling. The sections were then treated with a blocking agent (0.5% bovine serum albumin) for 45 minutes at room temperature after washing in PBS and then incubated overnight at 4°C with insulin antibody (1:1000 dilution) followed by the corresponding anti-guinea pig FITC-conjugated secondary antibody after washing in PBS. The sections were then incubated with glucagon antibody (prediluted) overnight at 4°C followed by anti-rabbit TRITCconjugated secondary antibody and then mounted using Immunomount® (Thermo electron Corporation, USA) and viewed with the Carl Zeiss fluorescent microscope.
8
Retrograde Labeling and Morphometric Analysis
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All slides were rinsed in PBS to remove the OCT and treated for lipofuscin reduction by incubating the tissue in 750 μM cupric sulfate/50 mM ammonium acetate for 40 minutes as reported elsewhere48 (link). The tissue was then rinsed in PBS and cover-slipped using immuno-mount (Fisher Scientific, Waltham, MA). The FG+ and FR+ cells in the spinal cord and DRG were identified with a Zeiss fluorescence microscope using a wide band ultraviolet (UV) excitation filter. To prevent double counting, only positive retrograde labeled cells with a distinct nucleolus were included. Positively labeled DRG were also counted and the area was analyzed using the analyze-particle option of Image J, processing software. The DRG soma area was categorized into small (<300 μm2), medium (300–700 μm2), and large (≤700 μm2). Labeled cell quantification was corrected by accounting for section thickness and split nuclei count using methods described by Abercrombie49 .
9
Immunofluorescence Staining for PI(4,5)P2
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Four-micrometer formalin-fixed, paraffin-embedded sections were de-paraffinized with xylene. Samples were hydrated through a descending alcohol series and endogenous peroxidases were inactivated by incubation in 3% H2O2 in methanol for 20 min. Antigen unmasking was performed by boiling the tissue sections in 10 mM citric buffer (pH6; for PI(4,5)P2 and H3K4me3 detection) for 3 min in a microwave followed by 15 min resting at RT. Blocking of unspecific antigen sites was achieved with 50% goat serum (Thermo Scientific, Schwerte, Germany) in PBS for 1h at RT. Incubation with primary antibody against PI(4,5)P2 was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P2 antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT. The fluorescence signals were generated with the ‘‘TSA™, Fluorescein System’’ (Perkin Elmer, Rodgau, Germany). After the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and embedded in Immunomount (Fisher Scientific, Schwerte) the specific signals were visualized by immunofluorescence microscopy using the Leica microscope DMI6000B, Leica camera DFC365FX and Leica Application Suite software v3.3.0.16799.
10
Immunofluorescence Staining of Neuronal Cells
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SN56 cells or mouse cortical neurons were fixed for 15 minutes with 4% paraformaldehyde (Alfa Aesar; Cat No. 43368). Cells were permeabilized for 5 minutes with 0.1% TritonX-100 in PBS and blocked with 2% BSA for 1 h. Cells were incubated with primary antibodies overnight at 4°C, washed twice with PBS, and stained with secondary antibody for 1 h. After staining, confocal plates were store at 4°C in PBS, and coverslips were mounted on glass slides with ImmunoMount (Fisher) and stored at 4°C.
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