Ovaries from 3 to 4 day old females were dissected in PBS and fixed for 20 min at room temperature (RT) in 4% paraformaldehyde in PBS, pre-absorbed in 2% BSA, 0.3% Tween-20 or Triton X-100 in PBS for 1 h at RT, incubated with primary antibodies overnight 4 °C, secondary antibodies for 2 h at RT, stained with 1 µg/ml Hoechst 33342 (Molecular Probes) for 10 min at RT, and ovarioles were separated and mounted in 80% glycerol 0.5% N-propylgalate mounting media. Antibody dilutions: mouse monoclonal anti-Lamin C (DSHB, LC28.26) 1:200, mouse monoclonal anti-Fasciclin 3 (DSHB, 7G10) 1:40, mouse monoclonal Eyes Absent (DSHB, eya10H6) 1:200, rabbit polyclonal anti-GFP (Life Technologies), anti-mouse-Cy3 (Life Technologies) 1:200, and anti-rabbit-Alexa Fluor 488 1:200 (Cell Signaling). To detect apoptosis, ovaries stained with Lamin C were fixed a second time (4% paraformaldehyde, PBS) prior to ApopTag® Red In situ Apoptosis Detection Kit (Milipore). Ovaries were incubated in equilibration buffer 10 s RT, 1 h 37 °C in Tdt enzyme solution, 10 min RT in stop/wash buffer, washed 0.3% Tween-20, PBS, and Rhodamine antibody solution for 30 min at RT. Confocal images were taken using a Zeiss 510Meta LSM or Zeiss 800 with multiple consecutive Z-sections (0.2–0.3 mm per slice), processed with ImageJ (1.46 R) and annotated using Adobe Photoshop CS5.1 and Adobe Illustrator CC 2015.
Alexa fluor 488 anti rabbit
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 488 anti-rabbit is a fluorescent secondary antibody specifically designed for the detection of rabbit primary antibodies. It is conjugated with the Alexa Fluor 488 fluorescent dye, which excites and emits light at specific wavelengths, allowing for the visualization and analysis of target proteins in various experimental applications.
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Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 anti mouse antibodies, by Cell Signaling Technology (2 mentions)
Illustrator cc 2015, by Adobe (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Anti mouse cy3, by Thermo Fisher Scientific (1 mentions)
Antispike, by Sino Biological (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Desmin, by Merck Group (1 mentions)
Vimentin, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
8 protocols using alexa fluor 488 anti rabbit
1
Immunofluorescence Imaging of Ovarian Development
2
Flow Cytometry Analysis of hACE2 Expression
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hACE2-gp130 cell surface expression of stably transfected Ba/F3-gp130 cells was detected by specific antibodies. A total of 5 × 105 cells were washed in fluorescence-activated cell sorter (FACS) buffer (PBS, 1% bovine serum albumin [BSA]) and then incubated in 50 μL containing the indicated primary antibody (anti-hACE2, catalog number AF933; Bio-Techne) (1:80) for 1 h at room temperature. Cells were washed and resuspended in 50 μL containing the secondary antibody (Northern Light 495-conjugated Fab anti-goat IgG, catalog number NL003; Bio-Techne) (1:100) and incubated for 1 h at room temperature. Cells were washed and resuspended in 500 μL of FACS buffer. A total of 20,000 cells were recorded and analyzed by flow cytometry (BD FACSCanto II flow cytometer using FACSDiva software; BD Biosciences). Data analysis was conducted using FlowJo version 10 (Tree Star Inc., USA).
Flow cytometry inhibition experiments were conducted with 5 × 105 Ba/F3-gp130-hACE2-gp130 cells in the presence of 5 nM S-RBD, inhibitory proteins, and the primary antibody (antispike, catalog number 40150-R007; Sino Biological) (1:50) for 1 h at room temperature. Cells were washed and resuspended in 50 μL containing a secondary antibody directed against antispike-IgG (anti-rabbit–Alexa Fluor 488; Cell Signaling Technology) (1:250) for 1 h at room temperature. Afterward, cells were treated as described above.
Flow cytometry inhibition experiments were conducted with 5 × 105 Ba/F3-gp130-hACE2-gp130 cells in the presence of 5 nM S-RBD, inhibitory proteins, and the primary antibody (antispike, catalog number 40150-R007; Sino Biological) (1:50) for 1 h at room temperature. Cells were washed and resuspended in 50 μL containing a secondary antibody directed against antispike-IgG (anti-rabbit–Alexa Fluor 488; Cell Signaling Technology) (1:250) for 1 h at room temperature. Afterward, cells were treated as described above.
3
Characterizing Peripheral Nerve Degeneration in Diabetic Neuropathy
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Formalin-fixed paraffin-embedded human nerve sections of participants with and without DPN following lower limb amputation were obtained from the Department of Vascular Surgery, Heidelberg University Hospital and the Tissue Bank of the National Center for Tumor Diseases, Heidelberg as described previously [18 (link)]. Sections from the sciatic nerve (5 μm) were deparaffinated and antigen retrieval was performed using Target Retrieval Solution (DAKO, USA), according to the manufacturer’s instructions. Then, 0.5% saponin was used to permeabilise and 10% donkey serum in PBS-0.2% Triton X100 was used for blocking. Following an overnight incubation at 4°C with anti-MPZ antibody (1:100; Abcam), the sections were washed and incubated with anti-rabbit Alexafluor488 (1:1000; Cell Signaling, USA) for 1 h at room temperature. The sections were then washed and stained with DAPI (Thermo Fischer). Fluorescent images were taken on a confocal laser-scanning microscope (A1R; Nikon, Japan) and analysed using ImageJ software. Participant characteristics are provided in ESM Table 2 .
4
Immunofluorescence Analysis of Formalin-Fixed Tissues
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Formalin-fixed, paraffin-embedded tissues were sectioned (5μm thickness), transferred to Superfrost/Plus Microscope Slides (Fisher Scientific), and incubated for 30’ at 60°C. Slides were washed with PBST and blocked with PBS + 5% fetal bovine serum (Sigma-Aldrich) and 3% goat serum (Sigma-Aldrich). Slides were then incubated for 1 hour at room temperature with antibodies specific for albumin (Santa Cruz Biotechnologies), vimentin (Millipore), desmin (Sigma-Aldrich), α-SMA (Sigma-Aldrich), or GFP (Invitrogen). They were then washed in PBST and incubated with combinations of the following secondary antibodies: Cy3 labeled goat anti-rabbit IgG (GE Healthcare), Cy3 labeled anti mouse and Cy3 anti-chicken (Millipore), or Alexa Fluor 488 anti-rabbit (Cell Signaling Technology). After washing in PBST, slides were counterstained with Hoechst 33258 (Molecular Probes) and mounted using Prolong Gold Antifade Reagent (Invitrogen).
5
Immunofluorescence Staining of SW480 Cells
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SW480 cells were seeded on cell slides in a 24-well plate and cultured for 24 h. After the medium was decanted, the cells were washed three times with cold PBS. Then, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 5 min. After the cells were washed three times with cold PBS, PBS containing 5% bovine serum albumin was used to block the samples for 1 h at room temperature. Then, 1:100 diluted indicated antibody in PBS was added to cover the cell slides and incubated overnight at 4ºC. After three washes with PBS, a 1:500 dilution of Alexa Fluor 488 anti-rabbit and a 1:1000 dilution of Alexa Fluor 647 anti-mouse antibodies (Cell Signal Technology) were added to cover the cell slides. Then, the cell nuclei were stained with DAPI for 1 h at room temperature. After five times wash with PBS, the cells on the slides were mounted by using ProLong Gold antifade reagent (Invitrogen), and images were acquired using a fluorescence microscope (Zeiss, Oberkochen, Germany).
6
Immunofluorescence Labeling of Glutaminase and Cdh1
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Free floating sections were washed in Tris buffered saline with Triton solution (0.1%) (TBS-Tx), 3 times at room temperature (RT). Sections were incubated for 3 h at RT in a pre-incubation solution of 2% bovine serum (BSA; A7906, Sigma) in TBS-Tx. After the pre-incubation, the tissue was incubated, 2 days at 4 °C, in a primary solution containing BSA (2%) in TBS-Tx with the primary antibodies: rabbit anti-Glutaminase (Proteintech; 1:200, Chicago, USA), mouse anti-Cdh1 (Abcam; 1:100, Cambridge, UK) or rabbit anti-Cdh1 (NBP2, Novusbio; 1:250, Cambridge, UK). Afterwards, sections were rinsed 2 times in TBS-Tx and 2 times in HEPES (10 mM) in physiological saline solution and then incubated for 3 h at RT in a solution of 2% BSA in HEPES (10 mM) with the secondary antibody: Alexa Fluor 488 anti-rabbit (CellSignaling Tech; 1:1000, Danvers, USA) and Alexa Fluor 647 anti-mouse (CellSignaling Tech; 1:1000). Finally, sections were rinsed in HEPES (10 mM), mounted onto pig skin gelatin-coated slides (0.5%) and covered with a drop of VECTASHIELD Mounting Medium with DAPI (H1200, Vector, Burlingame, USA) for nuclear staining and a drop of fluorescence mounting medium (S3023, DAKO, Barcelona, Spain) was added.
7
Isolation and Characterization of Cardiac Nuclei
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Nuclear isolation was performed as previously described (Bergmann and Jovinge, 2012 ) Briefly, approximately 50 mg of cardiac tissue was dissected with a scalpel, suspended in lysis buffer (0.32 M Sucrose, 10 mM Tris-HCL (pH 8), 5 mM CaCl2 5 mM Magnesium Acetate, 2 mM EDTA, 0.5 mM EGTA, 1mM DTT) and homogenized first using a Omni TH rotor-stator followed by eight strokes with a Dounce homogenizer. Tissue homogenate was then passed through 100 and 70 μm strainers (BD Biosciences), centrifuged at 700g for 10 min at 4 °C and resuspended in sucrose buffer (2.1 M Sucrose, 10 mM Tris-HCl (pH 8), 5 mM Magnesium Acetate, 1 mM DTT). The nuclear suspension was carefully layered on top of fresh sucrose buffer and centrifuged at 13,000g for 60 min at 4 °C. The pellet was dissolved in nuclei storage buffer (0.44 M Sucrose, 10 mM Tris-HCl (pH 7.2), 70 mM KCl, 10 mM MgCl2, 1.5 mM Spermine).
Isolated nuclei were stained with a PCM1 antibody (1:500, Sigma, HPA) for 1 h at 4 °C. Cells were washed twice with nuclei storage buffer and stained with a secondary antibody Alexa Fluor 488 anti rabbit (1:1000, Cell signaling) for 1 h at 4 °C and then stained with Draq5 (1:300, BioStatus, Shepshed, UK).
Nuclei were analysed by the flow cytometry sorter BD FACSAria III and were gated based on forward scatter, Draq5 and PCM1.
Isolated nuclei were stained with a PCM1 antibody (1:500, Sigma, HPA) for 1 h at 4 °C. Cells were washed twice with nuclei storage buffer and stained with a secondary antibody Alexa Fluor 488 anti rabbit (1:1000, Cell signaling) for 1 h at 4 °C and then stained with Draq5 (1:300, BioStatus, Shepshed, UK).
Nuclei were analysed by the flow cytometry sorter BD FACSAria III and were gated based on forward scatter, Draq5 and PCM1.
8
Immunofluorescence Staining of HepG2 Cells
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We grew HepG2 cells on cell slides inside a 24-well plate for 24 h. The medium was then decanted and the wells were washed three times with cold PBS. The cells were then fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 5 min. After washing three times with PBS, the cells were blocked for 1 h at 25 °C in PBS with 5% bovine serum albumin. The primary antibodies were diluted by 1:100 in PBS with 1% bovine serum albumin (antibody dilution buffer) and incubated overnight at 4 °C. After washing three times with PBS, Alexa Fluor 488 anti-rabbit and Alexa Fluor 647 anti-mouse antibodies (Cell Signaling, USA) were added to the antibody dilution buffer at 1:500 and 1:1,000 dilutions, respectively. We then added DAPI to the slides, and incubated them for 1 h at room temperature. After washing the slides five times with PBS, we mounted them using ProLong Gold antifade reagent (Invitrogen, USA). We acquired images using a Two-photon super-resolution point scanning confocal microscope (Nikon, Japan) and selected representative images for each sample.
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