Primary cortical neurons were dissected from the cortex of embryonic day (E) 16–E18 Sprague Dawley rats (Shanghai SIPPR BK Laboratory Animals Ltd, China) embryos as previously described41 (link). In brief, primary neurons were seeded onto coverslips previously coated with poly-D-lysine (PDL) in 24-well plates (150,000 cells/coverslip). At 8-day in vitro (DIV), neurons were treated with PBS and 100 nM (final concentration) WT1a or G51D α-syn PFFs, and collected for immunofluorescence at 10/14-day post-treatment. The primary antibodies used in the assay were the phospho-α-synuclein (S129) antibody (Abcam, cat. no. ab51253) and the MAP2 antibody (Abcam, cat. no. ab5392). The antibodies were diluted at a ratio of 1:1000. The intensity of confocal images was analyzed by Image J 2.0.0. The secondary antibodies included goat anti-rabbit IgG Alexa Fluor 568 (Abcam, cat. no. ab175471) and goat anti-chicken Alexa Fluor 488 (Thermo Fisher, cat. no. A-11039). All rat experiments were performed followed the protocols approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry (IRCBC), Chinese Academy of Sciences (CAS). There are three samples in each group.
Alexa fluor 568 goat anti rabbit igg
Manufactured by Abcam
Sourced in United Kingdom
Alexa Fluor 568 goat anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 568 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
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Lab products found in correlation
Wash buffer, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Map2 antibody, by Abcam (1 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Ab14140, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Click it plus edu alexa imaging kit, by Thermo Fisher Scientific (1 mentions)
Ab175471, by Abcam (1 mentions)
Fv1000 ix81, by Olympus (1 mentions)
Immunostaining permeabilization solution with saponin, by Beyotime (1 mentions)
Quickblock blocking buffer for immunol staining, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Immunol staining fix solution, by Beyotime (1 mentions)
Cm3050, by Leica camera (1 mentions)
Image pro premier software, by Media Cybernetics (1 mentions)
Anti cdx2 mouse monoclonal antibody, by BioGenex (1 mentions)
Alexa fluor 488 labelled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti ocln, by Thermo Fisher Scientific (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
12 protocols using alexa fluor 568 goat anti rabbit igg
1
Immunofluorescence analysis of α-synuclein PFFs in primary cortical neurons
2
Detecting EBV Latent Antigen in Epithelial Cells
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It was performed in order to demonstrate EBV latent antigen expression in epithelial cells. IHC for LMP2A was assessed as previously described, but LMP2A primary Ab was detected with rabbit anti-rat IgG FITC conjugated (Vector Labs). Subsequently, the same sections were subjected to antigen retrieval with 20 mM Tris/0.65 mM EDTA buffer pH 8 at 121 °C for 5 min. After blocking, sections were incubated with rabbit monoclonal anti-Cytokeratin 7 Ab (Ventana), and detected with goat anti-rabbit IgG (AlexaFluor®568, ab175471, Abcam) as secondary Ab. Finally, sections were stained with nuclear dye Höechst and covered using commercial aqueous mounting medium.
3
Asymmetric Cell Division in AML Cells
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AML cells were fixed with 4% paraformaldehyde, permeabilized with 1× wash buffer (Dako), and blocked with 10% normal goat serum (Invitrogen) in wash buffer (Dako). Cells were incubated overnight at 4°C with the primary rabbit αNumb antibody (ab14140; Abcam) diluted 1:50 in diluent (Dako). Incubation with the secondary antibody, goat anti–rabbit IgG–Alexa Fluor 568 (dilution 1:1,000; Abcam), was performed for 1 h at room temperature. DAPI (Roche) was used to counterstain for DNA. Samples were acquired on an ImageStreamX Mark II imaging flow cytometer and analyzed using INSPIRE and IDEAS software. Dividing cells were analyzed by IDEAS software in a blinded fashion by two independent researchers. A difference in Numb intensity between daughter cells of at least 1.8-fold was defined as asymmetric cell division according to Zimdahl et al. (2014) (link).
4
Immunofluorescence and EdU Labeling Assay
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Cells were blocked with bovine serum albumin (BSA) at 37°C for 30 minutes and then were incubated with CTNNB1 antibody (1:200, Rabbit monoclonal, C2206; Sigma‐Aldrich) at 4°C overnight, followed by Goat Anti‐Rabbit IgG (Alexa Fluor® 568, 1:1000; Abcam) for 1 hour at room temperature. The nuclei were counterstained with 4, 6‐diamidino‐2‐phenylindole (DAPI, 1:10000; 4084; Cell Signaling). For EdU labelling assay, cells isolated from spheres were treated with trypsin and accutase, and were seeded into chamber slides after being filtered into singled cell (Nunc; Thermo Scientific, Carlsbad, CA, USA; Figure S1 A). EdU intensity was detected after cells were incubated with 10uM EdU for staining 24 hours. Detection was performed using Click‐iT Plus EdU Alexa imaging Kit (MP 10637; Life Technologies, Carlsbad, CA, USA). The fluorescence images were obtained using an Olympus microscope, and the counting procedure was performed on twenty images that captured from each single experiment (Figure S1 B).
5
Immunofluorescence Microscopy of Transfected HEK293T Cells
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HEK293T cells were seeded in 24-well plates on coverslips, and after overnight culture indicated plasmids were transfected. At 24 h after transfection, cells were washed 3 times with cold PBS and then fixed with Immunol staining fix solution (Beyotime). The cells then were permeabilized with immunostaining permeabilization solution with saponin (Beyotime). After that, cells were blocked with QuickBlock blocking buffer for Immunol staining (Beyotime) and then incubated with primary antibody (C3956; anti-MYC; Sigma) at 4°C overnight. After washing 3 times with PBS, the cells were incubated with the secondary antibody (ab175471; goat anti-rabbit IgG Alexa Fluor 568; Abcam). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime). The cells were then analyzed with a laser confocal microscope (IX81-FV1000; Olympus).
6
Laminin Immunohistochemistry for Muscle Fiber Analysis
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The TA muscle samples were cut into 10 µm cross sections at -20°C with a cryostat (CM3050S, Leica, Germany) and mounted on silanized slides for immunohistochemical staining. After air-drying at room temperature and fixing with ice-cold 4% paraformaldehyde for 15 min, sections were blocked at room temperature for 1 h with 10% normal goat serum (NGS) and 1% Triton X-100 in PBS, then washed twice in PBS for 5 min. Next, sections were incubated in 5% NGS and 0.3% Triton X-100 in PBS for 16-20 h at 4°C with a primary antibody against laminin as a marker for basement membrane integrity (1:200 dilution, Abcam, Tokyo, Japan). The sections were washed several times with PBS, incubated with an appropriate secondary antibody (Alexa Fluor 568 goat anti-rabbit IgG, 1:500 dilution, Abcam), diluted with PBS containing 5% NS and 0.1% Triton X-100 for 1 h at room temperature, and finally mounted with Vectashield mounting medium. Images of TA muscle sections were obtained using a fluorescent light microscope (BX60; Olympus, Tokyo, Japan) and a charge-coupled device (CCD) camera (DP73; Olympus, Tokyo, Japan). Digital images at 200-fold magnification were used to determine the cross-sectional area of the muscle fibers (FCSA) in each TA muscle. The FCSAs of at least 100 fibers in each muscle were measured using Image-Pro Premier software (Media Cybernetics).
7
Evaluating Cloned Blastocyst Quality
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To evaluate the quality of the cloned blastocysts from FD somatic cells, cell numbers were examined using immunofluorescence staining as previously described12 (link). The primary antibodies used were an anti-CDX2 mouse monoclonal antibody (1:500; BioGenex, San Ramon, CA, USA, MU392A-UC) for detecting the TE cells and an anti-Nanog rabbit polyclonal antibody (1:500; Abcam, Cambridge, UK, ab80892) for detecting the ICM cells. The secondary antibodies used were Alexa Fluor 488-labelled goat anti-mouse IgG (1:500; Molecular Probes Inc., Oregon, USA, A11029) and Alexa Fluor 568 goat anti-rabbit IgG (1:500; Abcam, Cambridge, UK, A11036). DNA was stained with DAPI (2 μg/mL; Molecular Probes).
8
Caco-2 Cell Tight Junction Imaging
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Caco-2 cells were plated on 35-millimeter, collagen-coated glass-bottom dishes and were treated at 4 days post-confluence. After 24 h of stimulation, cells were fixed in methanol for 10 min at −20 °C, washed three times with high salt buffer, and incubated overnight at 4 °C with Anti-ZO1 (1:100, Cat. 402200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) or Anti-OCLN (1:100, Cat. 331588, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies in BSA 1X or BSA 1X with 0.1% saponin, respectively. For Anti-ZO1 Ab, cells were washed and treated with the secondary antibody Alexa Fluor® 568 Goat Anti-Rabbit (IgG) (1:100; Abcam, Cambridge, UK) at room temperature for 1 h. One micromolar of DAPI(Sigma-Aldrich, St Louis, MO, USA) was used to stain cell nuclei. Images were acquired by an SM710 inverted confocal laser scanning microscope (ZEISS, Oberkochen, Germany).
9
Immunoblotting Antibody Reagents for Signal Transduction
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Phospho AKT (Ser473, D9E) XP rabbit mAb, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody, HER2/ErbB2 antibody, and AKT (pan; 40D4) mouse mAb were purchased from Cell Signaling Technology (Danvers, MA). ERK 1 (C-19) antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX), and Anti-Pan-Ras (Ab-3) mouse mAb was from Sigma-Aldrich (St. Louis, MO). Anti-laminin 5 antibody and Alexa Fluor 568 goat anti-rabbit IgG were from Abcam (Cambridge, UK). Rictor mouse mAb 1G3P2C9 was obtained from Bethyl Laboratories (Montgomery, TX). Peroxidase AffiniPure goat anti-mouse IgG, peroxidase AffiniPure goat anti-rabbit IgG, and AMCA AffiniPure goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
10
Quantifying Pneumococcal Phagocytosis in THP-1 Cells
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Pneumococci attached to or phagocytosed by PMA-differentiated THP-1 cells were visualized by double immunofluorescence microscopy (DIF). Therefore, THP-1 cells (2 × 105) were seeded on sterile glass cover slips (12 mm, Hartenstein) and cultured at 37°C and 5% CO2, 72 h prior to the infection (in the presence of 200 nmol/ml PMA) and infected as described above. Post-infection, THP-1 cells were washed with infection medium to remove unbound bacteria and then fixed with 1% paraformaldehyde (Roth). After blocking with 5% bovine serum albumin (BSA, Roth), extracellular bacteria were stained using a polyclonal anti-pneumococcal serum (1:200) and secondary goat anti-rabbit IgG coupled to Alexa-Fluor-488 (1:500, Abcam). Intracellular pneumococci were stained with Alexa-Fluor-568 goat anti-rabbit IgG (1:500, Abcam) after permeabilization of the THP-1 cells with 0.1% Triton-X-100 (Sigma) (10 min, room temperature) and pneumococcal antiserum as primary antibody (1:200). For the statistical analysis 100 cells per experiment and time point were analyzed for the number of intracellular bacteria.
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