VEGF and p-VEGFR2 were examined on 8 μm cryosections (on day 7 after laser photocoagulation). The cryosections were blocked with 1% bovine serum albumin for 4 h at RT, then incubated with VEGF antibody (1:50) and p-VEGFR2 antibody (1:50) at 4 °C overnight. For VEGF and p-VEGFR2 staining, antigen retrieval was obtained through heated water bath at 37 °C for 10 min. Thereafter, the slides were stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; #A27034, Thermo Fisher Scientific), Alexa Fluor 546-conjugated goat anti-mouse IgG (1:200; #A11030, Thermo Fisher Scientific), and DAPI (1:500). The photomicrographs were taken by a digital high-sensitivity camera (Hamamatsu, ORCA-ER C4742-95, Japan).
Alexa fluor 546 conjugated goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 546-conjugated goat anti-mouse IgG is a secondary antibody used for detection and visualization of mouse immunoglobulin G (IgG) in various immunological assays. It is conjugated to the Alexa Fluor 546 fluorescent dye, which provides bright and photostable fluorescence.
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Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 488 conjugated donkey anti goat igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Orca er c4742 95, by Hamamatsu Photonics (1 mentions)
Jc 70a, by Abcam (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Permafluor aqueous mounting medium, by Thermo Fisher Scientific (1 mentions)
Sc 1851, by Santa Cruz Biotechnology (1 mentions)
Bz 9000, by Keyence (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
22 protocols using alexa fluor 546 conjugated goat anti mouse igg
1
Immunohistochemical Analysis of VEGF and p-VEGFR2
2
Quantifying Cell-Cell Interactions via Confocal Microscopy
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We performed a series of confocal microscopy image acquisition on 16-h co-cultured cells. Cells were fixed with a 10% formalin solution neutrally buffered (Sigma-Aldrich, St. Louis, MO, USA) at RT for 15 min and were permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells were then washed two times with PBS and were blocked in 0.5% bovine serum albumin (BSA) in PBS for 20 min before incubating overnight with the primary mouse monoclonal anti-CD31 antibody (1:500 clone JC/70A, AbCam, Cambridge, UK) and a mouse monoclonal anti-CD42b-FITC antibody (1:500; eBioscience, San Diego, CA, USA). The secondary antibody for the anti-CD31 was Alexa Fluor 546-conjugated goat anti-mouse IgG (1:1,000; Thermo Fisher Scientific, Waltham, MA, USA). About 4′,6-diaminophenyl-indole (DAPI) in ProLong® Gold Antifade Mountant (Thermo-Fisher, Waltham, MA, USA) was used for nucleic acid staining. Confocal images were obtained with a Nikon A1 MP confocal scanning system connected to an Eclipse T-i microscope, with an × 40 objective plus further 1 and × 3 magnification, acquired by Nis-Elements imaging software, and were analyzed by the processing Image-J/Fiji software (LOCI, University of Wisconsin-Madison, USA). The degree of co-localization was quantified using Mander's overlap coefficient (MOC). Data are presented as mean ± SEM with respect to untreated samples, from a minimum of 50 cells.
3
Immunofluorescence Staining of HCASMCs
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Immunofluorescence staining was performed as previously described.18 (link) In brief, HCASMCs were seeded on sterile coverslips that had previously been coated with 200 μL of 0.3% gelatin at room temperature for 30 minutes. After the cell confluence reached 90%–100%, the cells were preincubated with or without hesperetin for 30 minutes and then treated with SPC for 10 minutes. Next, the HCASMCs were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 2 minutes, blocked in NanoBio blocker solution (NanoBio Tech Co, Ltd, NY), and diluted in PBS for 60 minutes at room temperature. After that, the HCASMCs were stained with primary anti-ROCK (dilution time: 1:100; sc-1851, Santa Cruz, Dallas, TX) and anti-Fyn (dilution time: 1:100; 610164, BD Biosciences, NY) antibodies at 4°C overnight, followed by staining with Alexa Fluor 488-conjugated donkey antigoat IgG (A32814; Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 546-conjugated goat antimouse IgG (A11003; Thermo Fisher Scientific) secondary antibodies. Before observation, the cells were mounted with PermaFluor aqueous mounting medium (Thermo Fisher Scientific). The field of view was randomly selected to observe stained cells under an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan). Fluorescence intensity profile analysis was performed using Image J software.
4
Quantifying Autophagy in ARPE-19 Cells
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The ARPE-19 cells were cultured in 8-well chamber slides (SCS-N08, Matsunami Glass Ind., Ltd., Osaka, Japan). At the evaluation time points, the cells were washed with PBS, then fixed with 4% paraformaldehyde (Wako) for 15 min, blocked with 3% goat serum for 30 min, and then incubated overnight at 4 °C with the anti-LC3 rabbit monoclonal antibody (1:200, CST). After washing, the cells were incubated for 1 h with the secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, Thermo Fisher Scientific). The cells were washed again and then incubated overnight at 4 °C with anti-SQSTM1/p62 mouse monoclonal antibody (1:500, GeneTex). After washing, the cells were incubated for 1 h with the secondary antibody (Alexa Fluor 546-conjugated goat anti-mouse IgG, Thermo Fisher Scientific). The cells were then washed again and counter-stained with Hoechst 33342 (1:1000, Thermo Fisher Scientific). Images were acquired using a confocal fluorescence microscope (FLUOVIEW FV10i; Olympus, Tokyo, Japan). Autophagosomes and SQSTM1-positive speckles were detected using image-processing software, Image-J (developed by the National Institutes of Health).
5
Immunofluorescence Staining of T Cells
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T cells extracted from normal or ectopic endometrium were fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 15 min and rinsed three times with phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA). Immunofluorescence staining was conducted using a standard protocol as follows. Samples were incubated in blocking buffer (5% normal goat serum, 0.5% Triton X-100 in PBS) at RT for 1 h and then with primary antibodies at 4°C overnight. The samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (A-11008, Thermo Fisher Scientific) and Alexa Fluor 546-conjugated goat anti-mouse IgG (A-11030, Thermo Fisher Scientific) for 1 h. Images were obtained using an LSM 880 inverted confocal laser scanning microscope with an Airyscan detector (Zeiss). The primary antibodies utilized are listed in the key resources table .
6
Immunofluorescence Analysis of Cultured Cells
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Cultured cells were fixed with paraformaldehyde and then washed with PBST (PBS-Triton X100) containing normal goat serum (Thermo Fisher Scientific) or normal donkey serum (Thermo Fisher Scientific) at room temperature for 30 min to block non-specific antibody-binding sites. The cells were incubated with primary antibodies against alkaline phosphatase (Novus, Littleton, CO, USA), osteopontin (Rockland, Pottstown, PA, USA), CD73 (Santa Cruz Biotechnology, Dallas, TX, USA), and vinculin (Sigma-Aldrich). After washing with PBST, the cells were incubated with secondary antibodies, including Alexa Fluor®® 488-conjugated donkey anti-goat IgG (Thermo Fisher Science), Alexa Fluor®® 546-conjugated goat anti rabbit IgG (Thermo Fisher Science), and Alexa Fluor®® 546-conjugated goat anti-mouse IgG (Thermo Fisher Scientific). F-actin was visualized using Alexa Fluor®® 488-conjugated phalloidin (Thermo Fisher Scientific). Finally, the cells were washed with PBST and mounted with Prolong Gold antifade reagent with DAPI (Thermo Fisher Scientific). Fluorescent images were captured using a fluorescence microscope (Biozero; Keyence, Osaka, Japan) and processed using Adobe Photoshop 10.0.
7
Antibody Validation for Neurodegenerative Research
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The antibodies used were as follows: rabbit anti-mouse SNX27 (1:50, Thermo Fisher Scientific, #23025), rabbit anti-mouse Glial Fibrillary Acidic Protein, GFAP (1:500, WAKO, #Z0334), rabbit anti-mouse Ionized calcium-binding adapter molecule 1, Iba1 (1:250, DAKO, #019-19741), rabbit anti-mouse cleaved-caspase3 (1:800, Cell Signaling Technology, #9664S), mouse anti-mouse NeuN (1:400, EMD Millipore, #MAB377), rabbit anti-mouse β-actin (1:5,000, proteintech, #20536-1-AP), Alexa-fluor-488-conjugated goat anti-rabbit IgG (1:500, Thermo Fisher Scientific, #11034), Alexa-fluor-546-conjugated goat anti-mouse IgG (1:500, Thermo Fisher Scientific, #11081), HRP-conjugated goat anti-rabbit IgG (H + L) (1:10,000, Thermo Fisher Scientific, #31460), and HRP-conjugated goat anti-mouse IgG (H +L) (1:10 000, Thermo Fisher Scientific, #31430).
8
Immunofluorescence Analysis of Malaria Sporozoites
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Sporozoites were collected from salivary glands at days 19 to 22 post-feeding and seeded on eight-well multi-well slides, then air-dried and fixed with cold acetone for 3 minutes. The slides were blocked with Blocking One Histo (nacalai tesque) at 37°C for 30 minutes and incubated with primary antibodies (1:2,000 for anti-RON4 rabbit polyclonal antibodies, and anti-circumsporozoite protein (CSP) mouse monoclonal antibodies; MRA-100, obtained through BEI Resources, NIAID, NIH) in PBST containing 5% Blocking One Histo at 37°C for 2 hours, followed by Alexa Fluor 488–conjugated goat anti-rabbit IgG and Alexa Fluor 546–conjugated goat anti-mouse IgG (1:500, Thermo Fisher Scientific) at 37°C for 30 minutes. Nuclei were stained with 2 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI). The samples were mounted in ProLong Gold antifade reagent (Thermo Fisher Scientific) and observed with an inverted fluorescence microscope (Axio Observer Z1; Carl Zeiss, Oberkochen, Germany).
9
Immunofluorescence Assay for Primary Cilia
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The cells grown on Lab‐Tek plates or coverslips were fixed with 3.7% formaldehyde in PBS for 15 min. After two washes with PBS, the cells were permeabilized with 0.1% Triton X‐100, transferred into a blocking solution (20% goat serum in PBS) for 30 min, and incubated with mouse anti‐acetylated tubulin (Ac‐tub) (T7451; Sigma‐Aldrich; 1:2000) and rabbit anti‐GFP (598; MBL; 1:2000) primary antibodies for 16–24 h at 4°C. The bound antibodies were detected using appropriate secondary antibodies (Alexa Fluor 546‐conjugated goat anti‐mouse IgG or Alexa Fluor 488‐conjugated goat anti‐rabbit IgG; Life Technologies Co.).45 (link) Because primary cilium in vitro cell culture is usually lying flat along the coverslip, the length from a two‐dimensional image was measured using a line measurement tool PhotoRuler Ver. 1.1 software (The Genus Inocybe, Hyogo, Japan) under the BZ‐9000 fluorescence microscope (Keyence). Data for at least 100 cilia per treatment were obtained from at least three independent experiments, and the values are presented as means ± SEM.
10
Quantification of Parkin Recruitment to Mitochondria
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HEK293 cells stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP were transfected with pCMV6-mCherry-Parkin (PARK2) and treated with 10 µM CCCP together with 100 nM Bafilomycin A1 for two hours on the next day, as described above. Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 20 min followed by a blocking step with 4% BSA/0.05% saponin for 30 min at room temperature. Cells were co-stained with primary antibodies against the mitochondrial marker MTC02 (abcam, ab3298; 1:500), and α-Tubulin/TUBA1B (Acris antibodies, SM568P; 1:500) overnight at 4°C. Secondary antibodies (Alexa Fluor 546-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rat IgG) were from Life Technologies and used at a dilution of 1:500 for one hour at room temperature. Analyzes were performed with a LSM510-Meta confocal microscope (Zeiss) equipped with 40/1.3 immersion objectives and emission wavelengths of 468 nm, 488 nm, 543 nm, and 633 nm. Quantification of mCherry-Parkin dots was performed based on the mitochondrial content (MTC02 signal) using ImageJ software v1.49k and a specific macro (Suppl. Material & Methods ).
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