Samples were fixed in 4% paraformaldehyde followed by permeabilization using 0.1% saponin. Cells were then incubated with anti-LDLR (1:100 dilution; BioVision) overnight. Alexa Fluor 488-conjugated anti-rabbit (1:200 dilution; 715–546–150 Jackson ImmunoResearch, Pennsylvania) was used for visualization. DAPI (ThermoFischer Scientific) was used to stain DNA. All pictures were taken with a Zeiss LSM 700 confocal system.
Alexa fluor 488 conjugated anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488-conjugated anti-rabbit is a fluorescently-labeled secondary antibody. It is designed to detect and visualize rabbit primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated anti mouse, by Jackson ImmunoResearch (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Ab128919, by Novus Biologicals (2 mentions)
Alexafluor 594 conjugated anti rabbit, by Jackson ImmunoResearch (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700 confocal system, by Zeiss (1 mentions)
Ab4448, by Abcam (1 mentions)
Ab191114, by Abcam (1 mentions)
Ab45688, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Ti2 e inverted microscope, by Nikon (1 mentions)
Plan apo objective, by Nikon (1 mentions)
Ds qi2 monochrome cmos camera, by Nikon (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Anti ha h6908, by Merck Group (1 mentions)
Cytospin cytocentrifuge, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Rabbit anti s100b, by Agilent Technologies (1 mentions)
Anti cd31, by BioLegend (1 mentions)
10 protocols using alexa fluor 488 conjugated anti rabbit
1
Quantifying LDLR Expression by Immunofluorescence
2
Centrosome Immunofluorescence in Hepatocytes
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Sorted primary hepatocytes were plated on collagen-coated chamber slides, and incubated overnight at 37 °C. Then, cells were fixed with methanol and immunostained with rabbit anti-Pericentrin (1:1000, abcam, ab4448), Alexa Fluor 488-conjugated anti-rabbit (1:200, Jackson ImmunoResearch), and Alexa Fluor 647-conjugated anti-gamma Tubulin (1:1000, abcam, ab191114). To eliminate false positive signals from nonspecific immunostaining, centrosomes were identified as structures positive for both Pericentrin and gamma Tubulin. Nuclei were stained with Hoechst, and the nuclear number in each cell was evaluated on microscopy.
3
TGFβ-Induced Fibronectin Expression in LX-2 Cells
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LX-2 cells were cultured for 3 days in the presence or absence of TGFβ (Peprotech, catalog no. 100-21). Human ILT3ECD-Fc (5 μg/mL) was added for 30 minutes at room temperature, and the cells were fixed with 4% paraformaldehyde for 10 minutes. In some experiments, Fc-tagged human B7-H4 ECD (B7-H4-Fc; R&D Systems, catalog no. 8870-B7) was used as a negative control for LX-2 cell binding. Cells were blocked (10% FBS, 1% BSA, 0.01% NaN3 in PBS) at room temperature for 1 hour, then incubated with rabbit anti-fibronectin clone F14, which is cross-reactive to human and mouse fibronectin (Abcam, catalog no.: ab45688, RRID: AB_732380), at a 1:500 dilution for 1 hour at room temperature. Cells were then washed and stained with secondary antibodies, either AlexaFluor 488–conjugated anti-rabbit (Jackson ImmunoResearch, catalog no. 111-546-046, RRID: AB_2338055, 1:1,000 dilution) or AlexaFluor 647–conjugated anti-human (Jackson ImmunoResearch, catalog no. 109-606-098, RRID: AB_2337899, 1:500 dilution), for 1 hour at room temperature. Nuclei were stained with Hoechst (Thermo Fisher Scientific, catalog no. PI62249, 1:10,000 dilution) for 10 minutes at room temperature. Cells were imaged using a Nikon Ti2-E inverted microscope equipped with a 20 × 0.75 NA Plan Apo objective (Nikon), a DS-Qi2 monochrome CMOS camera (Nikon) and a Sola SE II 360 Light Engine (Lumencor).
4
Immunofluorescence Microscopy of Hematopoietic Cells
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5 × 105 cells were adhered to microscope slides using a Cytospin cytocentrifuge (Thermo Fisher Scientific) for 3 min at 200g and fixed in 4% formaldehyde (Pierce) for 15 min. Cells were permeabilised in 0.1% Triton X-100 and nonspecific staining was prevented by incubation in 3% bovine serum albumin. Antibodies were applied for 1 h at room temperature before washing, anti-HA (H6908; Sigma-Aldrich) at 1:200, anti-EVI1 (2593; Cell Signalling Technology) at 1:200, anti-RUNX1 (C-terminal, sc-28679 H-65; Santa Cruz Biotechnology) at 1:200 or anti-CBFβ (sc-56751; Santa Cruz Biotechnology), and secondary Alexa Fluor 488–conjugated anti-rabbit (Jackson ImmunoResearch) at 1:200. Slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Slides were visualised using a Zeiss LSM 780 equipped with a Quasar spectral (GaAsP) detection system, using a Plan Achromat 40× 1.2 NA water immersion objective, Lasos 30 mW Diode 405 nm, Lasos 25 mW LGN30001 Argon 488, and Lasos 2 mW HeNe 594 nm laser lines. Images were acquired using Zen black version 2.1. Post-acquisition brightness and contrast adjustment was performed uniformly across the entire image.
5
Immunofluorescent Analysis of Uveal Tumors
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Formalin-fixed paraffin-embedded uveal tumor sections (10 μm) were immunolabeled with Rabbit anti-S100B (DAKO) and anti-CD31 (Biolegend) antibodies to identify melanoma cells and blood endothelial cells, respectively, as described previously [16 (link)]. For analysis of proliferating cells, blood and LVs in uveal tumors, cryosections were stained with rat anti-Ki67-biotinalyted (eBioscience), armenian hamster anti-CD31 (PECAM-1, Millipore) or rat anti-CD31 (PECAM-1, BD Biosciences) and rabbit anti-Lyve-1 (Abcam) antibodies. Cy3-conjugated streptavidin, Alexafluor647-conjugated or Cy3-conjugated anti-armenian hamster and Alexafluor488-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories) antibodies were used for detection. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for cell nuclei visualization and mounted for analysis. Immunofluorescent labelled specimens were viewed with a fluorescence wide field (Axio Imager.Z1, Axioxam HRM camera; Carl Zeiss Micro Imaging, Jena, Germany). Tumor areas and BV size were measured using ImageJ software.
6
Epigenetic Modifications in Embryogenesis
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The embryos at the defined time points after fertilization were fixed by 4% polyformaldehyde overnight at 4°C. Then, they were dechorionated manually and dehydrated with methanol. The whole-mount IFs with Dnmt1 antibody (Santa Cruz Biotechnology, catalog no. sc-20701), 5mC antibody (Abcam, catalog no. ab10805), and pH2AX antibody [Cell Signaling Technology, catalog no. 2577S], were done with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, catalog no. D1306) staining and performed as previously described (20 (link)). The secondary antibodies were Alexa Fluor 488–conjugated anti-rabbit and Alexa Fluor 488–conjugated anti-mouse (Jackson ImmunoResearch; 1:200 dilution). After staining, embryos were deyolked by tweezers and mounted on glass slides in mounting medium (Sigma-Aldrich, catalog no. P3130) at animal polar upturned position. Images were acquired on 710 or 880 META laser scanning confocal microscope and manipulated by ZEN software. Treated or untreated embryos were anesthetized at desired stages with 0.02% tricaine and mounted in 5% methyl cellulose (Sigma-Aldrich, catalog no. M-6385) for observation, and phenotype pictures were taken under Nikon SMZ1500 microscope.
7
Immunostaining of Mitotic Proteins
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Primary antibodies used for immunostaining were anti-TRF1 (Abcam, ab192629, 1:100), anti-BubR1 (Abcam, ab28193, 1:100), anti-Zw10 (Abcam, ab21582, 1:100), anti-acetylated-α-tubulin (Sigma Aldrich, T7451, 1:500; Abcam, ab179484, 1:500), anti-centromere (Antibodies Incorporated, 15-234, 1:100), anti-CENP-A (Cell Signaling, #2048, 1:100), anti-Survivin (Cell Signaling, #2808, 1:100), anti-Hec1 (Santa Cruz Biotechnology, sc-515550, 1:100), anti-H3T3ph (Upstate, 07-424, 1:100), anti-SMC3 (Abcam, ab128919, 1:100), anti-SMC4 (Novus Biologicals, NBP1-86635, 1:100), and anti-TRF2 (Abcam, ab13579, 1:100). Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-mouse (Jackson ImmunoResearch, 111-585-146, 1:500), Alexa Fluor 488-conjugated anti-rabbit (Jackson ImmunoResearch, 115-545-144 1:500), Alexa Fluor 594-conjugated anti-rabbit (Jackson ImmunoResearch, 111-585-144, 1:500), and Alexa Fluor 488-conjugated anti-sheep antibodies (Abcam, ab150177, 1:500).
8
Mambalgin-2 Targets in Mel P Cells
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To study the mambalgin-2 molecular targets in mel P cells, the cells were seeded on round glasses in 24-well plates (15 × 104 cells/well) in the acidic medium and grown for 24 h. Thereafter, cells were fixed with 4% PFA for 15 min, blocked by 2% bovine serum albumin solution in PBS for 1 h and incubated with the primary antibodies for ASIC1a (sheep, 1:500, ABIN350049, Antibodies-Online), α-ENaC (rabbit, 1:500, ABIN1841945, Antibodies-Online) or γ-ENaC (mouse, 1:500, ABIN1865926, Antibodies-Online) subunits. After that, cells were incubated with the TRITC-conjugated anti-sheep (1:500, 713-025-003, Jackson Immunoresearch), AlexaFluor488-conjugated anti-rabbit (1:500, 611-545-215, Jackson Immunoresearch) or AlexaFluor488-conjugated anti-mouse (1:500, 715-545-150, Jackson Immunoresearch) antibodies and with mambalgin-2 labeled by CF647 dye. Cell nuclei were stained by Hoechst 33342, cells were mounted in the ProLong Gold antifade mounting medium (Life Technologies) and observed using the Carl Zeiss LSM710 inverted confocal microscope (Carl Zeiss, Jena, Germany) using × 60 (1.4) oil-immersion objective.
9
Immunofluorescence Staining of Mitotic Markers
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The following primary antibodies were used: anti‐γ‐H2AX (Abcam, ab22551, 1:250), anti‐MDC1 (Abcam, ab241048, 1:100), anti‐acetylated‐α‐tubulin (Sigma‐Aldrich, T7451, 1:500; Abcam, ab179484, 1:500), anti‐Mad2 (Abcam, ab70383, 1:100), anti‐BubR1 (Abcam, ab28193, 1:100), anti‐centromere (ACA; Antibodies Incorporated, 15–234, 1:100), anti‐SMC3 (Abcam, ab128919, 1:100), anti‐SMC4 (Novus Biologicals, NBP1‐86635, 1:100) and anti‐CENP‐A (Cell Signaling, #2048, 1:250). Alexa Fluor 488‐conjugated anti‐mouse (Jackson ImmunoResearch, 115‐545‐144, 1:500), Alexa Fluor 488‐conjugated anti‐rabbit (Jackson ImmunoResearch, 115‐545‐144 1:500), Alexa Fluor 594‐conjugated anti‐rabbit (Jackson ImmunoResearch, 111‐585‐144, 1:500) and Rhodamine (TRITC) ‐conjugated anti‐human (Jackson ImmunoResearch, 109‐025‐088, 1:100) were used as secondary antibodies.
10
Lineage Tracing of Tbx3+ Cells
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Livers and plantar skin tissues from 6-week-old Rosa26CAG-LSL-NuM-mCherry;Tbx3CreERT2 mice that had been administered tamoxifen or corn oil were collected and cryoprotected in PBS containing 20% sucrose and frozen in optimal cutting temperature compound. The samples were sectioned, immunostained, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in Tris-buffered saline for 15 min at room temperature. Then, the sections were blocked with Blocking-One Histo (Nacalai Tesque) at room temperature for 1 h, incubated with primary antibodies at 4 °C overnight, washed, and incubated for 1 h with secondary antibodies. The samples were mounted using VECTASHIELD PLUS Antifade Mounting Medium with DAPI (Vector Laboratories). The primary antibodies were anti-Tbx3 (rabbit, 1:200, ab99302; Abcam), anti-mCherry (chicken, 1:1000, ab205402; Abcam), anti-HNF4a (goat, 1:500, sc-6556; Santa Cruz Biotechnology), and anti-CD49f (rat, 1:500, 313602; Biolegend). The secondary antibodies were Alexa Fluor 488-conjugated anti-rabbit, Cy3-conjugated anti-chicken, Alexa647-conjugated anti-mouse, and Alexa647-conjugated anti-rat (Jackson ImmunoResearch, West Grove, PA, USA). All images were acquired using an Olympus FV3000 confocal microscope.
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