Subconfluent SH-SY5Y cells grown on glass coverslips were exposed for 48 h to 5 μM (monomer concentration) α-synuclein aggregates grown in the presence or in the absence of cowpea extract at different molar ratios (1:0.5, E0.5; 1:1, E1). Cell membrane labelling was performed by incubating the cells with 10 ng/ml Alexa Fluor 488-conjugated CTX-B (Cholera toxin B-subunit) in cold complete medium for 30 min at room temperature. Then, cells were fixed in 2.0% buffered paraformaldehyde for 6 min and permeabilized by treatment with a 1:1 acetone/ethanol solution for 4 min at room temperature, washed with PBS and blocked with PBS containing 0.5% BSA and 0.2% gelatin. After incubation for 1 h at room temperature with rabbit anti-synuclein polyclonal antibody (1:600 in blocking solution), the cells were washed with PBS for 30 min under stirring and then incubated with Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Molecular Probes) diluted 1:100 in PBS. Finally, cells were washed twice in PBS and once in distilled water to remove non-specifically bound antibodies. Digital images were taken with a confocal Leica TCS SP8 scanning microscope (Leica, Mannheim, Ge) equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica HC PL Apo CS2 X63 oil immersion objective.
Alexa fluor 568 conjugated anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor® 568-conjugated anti-rabbit secondary antibody is a fluorescent-labeled detection reagent used in immunoassays and immunohistochemistry. It specifically binds to rabbit primary antibodies, allowing visualization and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Boston BioProducts (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Antibody against nf κb2 p100 52, by Cell Signaling Technology (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Primary antibody against ha, by Roche (2 mentions)
Goat serum, by Nichirei Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Ab138501, by Abcam (1 mentions)
Triton x, by Merck Group (1 mentions)
Nunc lab tek, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Aurora b, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Ab3526, by Abcam (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
10 protocols using alexa fluor 568 conjugated anti rabbit secondary antibody
1
Visualizing α-Synuclein Aggregation Impact
2
Quantifying NF-κB2 p52 Localization
3
Quantifying Nuclear NF-κB2 Localization
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A549, H1650 and H226 cells were fixed in 4% paraformaldehyde (Boston Bioproducts), permeabilized in cold methanol (Fisher) and immunofluorescence staining was performed using an antibody against NF-κB2 p100/52 (Cell Signaling Technology; cat no.: 3017). The cells were then washed with PBS and incubated with Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Molecular Probes; cat no.: A11036). All images were captured by confocal microscopy (Nikon; 60× objective, 1.27 NA) and processed using ImageJ software (Version 1.6.0_24; https://imagej.nih.gov/ij ). The 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei were segmented manually by drawing a circular region of interest. Nuclear colocalization of NF-κB2 p52 (red fluorescent signal) with DAPI was measured. The mean nuclear fluorescence was calculated and plotted in GraphPad Prism software (Version 7).
4
Immunohistochemical Analysis of Itm2a Expression
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Serial frontal sections of the paraffin-embedded samples were cut at a thickness of 6-µm, and were mounted on silane-coated glass slides (Muto). The sections were de-waxed in xylene and re-hydrated in descending concentrations of ethanol. The slides were treated with antigen retrieval buffer (10 mM sodium citrate buffer, pH6.0) and microwaved for 5 min, and 10% goat serum (Nichirei Bioscience, Tokyo, Japan) was used to block non-specific immunoreactions. The specimens were incubated with the primary antibody, rabbit polyclonal anti-Itm2a antibody (1∶1000, 18306-1-AP; Proteintech Group, Inc., Chicago, IL) at 4°C. The primary antibodies against Itm2a were visualized using an Alexa Fluor 568-conjugated anti-rabbit secondary antibody (1∶2000, A11011; Invitrogen, Carlsbad, CA). Finally, the immunostained sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI, Dojindo, Kumamoto, Japan). The sections were rinsed in PBS three times at every interval between the steps.
The immunoreactivity of hepatocytes (Fig. S2 ) and chondrocytes was used as a positive control to develop the immunohistochemical staining procedures based on the manufacturer’s instructions and previous studies [18] (link), [20] (link), [31] (link).
The immunoreactivity of hepatocytes (
5
Immunofluorescent Staining and C-dot Internalization in SH-SY5Y Cells
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After treatment, α-Syn-EGFP expressing SH-SY5Y cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After permeabilization with 0.3% Triton-X (Sigma-Aldrich) in PBS for 5 minutes and saturation with 5% FBS in PBS for 1 hour at room temperature, fixed cells were incubated with an anti-α-Syn MJFR1 (ab138501, Abcam) primary antibody overnight at 4 °C. After incubation with an Alexa Fluor-568-conjugated anti-rabbit secondary antibody (Invitrogen) for 1 hour at room temperature, nuclei were stained with Hoechst 33 258 (Invitrogen) 1 : 2000 in PBS for 10 minutes, following mounting of coverslips on glass slides with Mowiol. Cell imaging was performed on a Leica 5000B epifluorescence microscope.
For the internalization study of C-dots, cells were seeded on 0.8 cm2 micro-slides (Nunc™ Lab-Tek™, ThermoFisher Scientific, USA) at a density of 2 × 104 cells per well and maintained overnight. For monitoring the penetration of the Lys C-dots into the cells, the SH-SY5Y cells were incubated with 2.5 mg mL−1 C-dots. Following 20 h of incubation of the cells with the C-dots, images of live cells were acquired on a Zeiss LSM880 confocal microscope (Germany), using a CLSM plan-Apochromat, ×20/0.8 M27 objective or ×60/1.35 numerical aperture oiled-immersion objective. Excitation/emission wavelengths were 405/561 nm.
For the internalization study of C-dots, cells were seeded on 0.8 cm2 micro-slides (Nunc™ Lab-Tek™, ThermoFisher Scientific, USA) at a density of 2 × 104 cells per well and maintained overnight. For monitoring the penetration of the Lys C-dots into the cells, the SH-SY5Y cells were incubated with 2.5 mg mL−1 C-dots. Following 20 h of incubation of the cells with the C-dots, images of live cells were acquired on a Zeiss LSM880 confocal microscope (Germany), using a CLSM plan-Apochromat, ×20/0.8 M27 objective or ×60/1.35 numerical aperture oiled-immersion objective. Excitation/emission wavelengths were 405/561 nm.
6
Visualization of HPV16 E6 and E7 Localization
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This assay is carried out as described previously [30 (link)]. Approximately 2 × 105 U-2 OS cells were plated onto coverslips. The cells were either mock-transfected or transfected with HA-tagged 16 E6 or E7. Cells were fixed with ice-cold absolute methanol 24 h after transfection and incubated with primary antibody against HA (Roche) and Aurora B (Cell Signaling), followed by Alexa Fluor® 568-conjugated anti-rabbit secondary antibody and Alexa Fluor®488-conjugated anti-mouse secondary antibodies (ThermoFisher Scientific), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The subcellular location of HPV16 E6 or E7 and AurB were examined under a fluorescence microscope (Leica, Wetzlar, Germany).
7
Subcellular Localization of HPV16 E6 and AurA
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This assay is carried out as described previously (29 (link)). Approximately 2 × 105 U-2 OS cells were plated onto coverslips. The cells were transfected with HA-tagged 16 E6 and Flag-tagged AurA. Cells were fixed with ice-cold absolute methanol 24 h after transfection and incubated with primary antibody against HA (Roche), Flag (Santa Cruz Biotechnology), and AurA (Cell Signaling), followed by Alexa Fluor 568-conjugated anti-rabbit secondary antibody and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (Thermo Fisher Scientific), and counterstained in 4′,6-diamidino-2-phenylindole (DAPI). The subcellular location of HPV16 E6 and AurA were examined under a fluorescence microscope (Nikon).
8
Immunofluorescence Analysis of ATF4 in L. amazonensis Infection
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RAW 264.7 cells (2 × 105) were plated in a 24 well-plate and infected for 8 hours with L. amazonensis. After infection, cells were fixed for 10 minutes with 4% paraformaldehyde and processed for immunofluorescence as follows: cells were permeabilized with 50 μg/mL digitonin in PBS for 5 minutes at room temperature, blocked with 1% bovine serum albumin (BSA, SIGMA-ALDRICH) in PBS for 1 hour at room temperature and then incubated overnight with anti-rabbit ATF4 polyclonal antibody (Cell Signaling) followed by incubation with an AlexaFluor 568-conjugated anti-rabbit secondary antibody (Thermo Scientific). DAPI was used for staining host and parasite DNA. Images were acquired with an LSM 780 multiphoton microscope and processed using ICY and ImageJ software.
9
Visualizing HPV58 E7 Localization
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U2‐OS cells plated onto coverslips were transfected with HPV58 E7. Cells were fixed with cold absolute methanol 24 hours after transfection and blocked with 3% bovine serum albumin. Cells were then probed with specified primary antibody against HPV58 E7 (ThermoFisher Scientific), followed by Alexa Fluor® 568‐conjugated anti‐rabbit secondary antibody (ThermoFisher Scientific) and counterstained in 4′,6‐diamidino‐2‐phenylindole (DAPI). The subcellular location of HPV58 E7 was examined under a confocal microscope (Leica, Wetzlar, Germany).
10
Adipocyte Area Analysis Protocol
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Adipose tissues were fixed with 4% formaldehyde in PBS and embedded in paraffin. Sectioned slides were stained with H&E. The adipocyte area was analyzed using ImageJ software (NIH, Bethesda, MD, USA). For immunofluorescence staining of perilipin, sections were permeabilized with 0.1% Triton X-100 for 15 min at room temperature and incubated with an antibody specific for perilipin A (ab3526, Abcam, Cambridge, MA, USA) in zymogen Ab diluent solution overnight at 4 °C prior to incubation with an Alexa Fluor-568-conjugated anti-rabbit secondary antibody (A11011, Thermo Fisher Scientific, Waltham, MA, USA). Samples were visualized with a confocal microscope (FluoView™ FV1000; Olympus, Tokyo, Japan).
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