Cells seeded on confocal dish were transfected with 1μg indicated plasmids. At 24h after transfection, cells were washed three times with PBS and fixed with 3.7% formaldehyde in PBS for 30min, then permeabilized by 1% Triton X-100 in PBS for 20min. After blocking with 5% BSA in PBS for 60 min, cells were incubated with mouse anti-FLAG antibody and anti-cleaved-caspase-3 diluted in 1:100 in 5% BSA in PBS at 4°C overnight. After washing three times with PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) (abcam) and Cy3 Goat Anti-Rabbit IgG (H+L) (ABclonal) diluted in 1:500 in 5% BSA in PBS for 30 min. After washing cells three times with PBS, cell nuclei were stained with DAPI (Invitrogen) diluted in 1:250 in PBS for 5 min. Fluorescent signals were detected by using a Nikon microscope and images were analyzed with the NIS-Elements Viewer 4.50.
Goat anti mouse igg h l alexa fluor 488
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) is a secondary antibody that binds to the heavy and light chains of mouse immunoglobulin G (IgG). It is conjugated to the Alexa Fluor® 488 fluorescent dye, which emits green fluorescence upon excitation.
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Lab products found in correlation
Goat anti rabbit igg h l alexa fluor 594, by Abcam (9 mentions)
Goat anti rabbit igg h l alexa fluor 647, by Abcam (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (7 mentions)
Ab150113, by Abcam (6 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (5 mentions)
Ab150117, by Abcam (4 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Fluorescence microscopy, by Olympus (3 mentions)
In cell analyzer 2500, by GE Healthcare (3 mentions)
Paraformaldehyde (pfa), by Sangon (3 mentions)
Goat anti rabbit igg h l alexa fluor 555, by Abcam (3 mentions)
Mouse monoclonal anti cd31, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Fluorescence microscope, by Leica camera (2 mentions)
Goat anti mouse igg h l alexa fluor 647, by Abcam (2 mentions)
Anti nlrp3, by Bioss Antibodies (2 mentions)
Antifading medium, by Solarbio (2 mentions)
77 protocols using goat anti mouse igg h l alexa fluor 488
1
Immunofluorescent Analysis of Apoptosis Signaling
2
Molecular Mechanisms of Cell Stress Response
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Primary antibodies applied in this study include antibodies against ORMDL3 (Abcam, UK, ab211522), Bax, Bcl-2, PARP, Caspase-3, Caspase-9, P62, Beclin1 (Proteintech-Group, Wuhan, China, 50599-2, 12789-1, 1337-1, 19677-1, 10380-1, 66184-1, 11306-1), LC3B (Novus-Biologicals, USA, NB100-2220), ATF4 (Cell Signaling Technology, D4B8, Danvers, USA), PERK, Phospho-PERK (Bioworld Technology, Shanghai, China, BS2156, BS66100), Goat anti-Rabbit IgG-HRP H&L, Goat anti-Mouse IgG-HRP H&L (Bioworld Technology, Shanghai, China, BS13279, BS12478), Alexa Fluor®488 Goat Anti-Rabbit IgG H&L, Alexa Fluor®488 Goat Anti Mouse IgG H&L(Abcam, UK, ab150077, ab150113). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), BCA Protein Assay Kit, BeyoECL Plus, DAPI Staining Solution, Apoptosis and necrosis Assay Kit, Annexin V-FITC/PI Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit, and ATP detection Kit were purchased from Biyuntian Institute of Biotechnology (Nanjing, China). MitoSOX Red Mitochondrial Superoxide Indicator was purchased from ThermoFisher Scientific (Shanghai, China).
3
Pronephros Cell Isolation and Flow Cytometry
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Salmon pronephros cells were isolated and processed for flow cytometry analysis (pronephros of each condition WTR control, control mock, fever, and no fever). Cell isolation was performed by disaggregating tissue through Falcon® cell strainers (100 µm) with DMEM media plus 10% FBS and 1% glutamax. The cells were pelleted by centrifugation at 1,200 rpm for 10 min. Maisey et al. (48 (link)) methodology was carried out with some modifications. In brief, pronephros cell was re-suspended and blocked with PBS plus 2% FBS immunofluorescence (IF) media. Cells were incubate for 1 h at 4°C with primary antibody anti-CD4-1 (protein G- or affinity-purified Abs) (49 (link)). Then, cells were suspended again with IF media containing secondary antibody BV421 Goat Anti-Rabbit IgG Clone Polyclonal (565014, BD Bioscience, NJ, USA) and Alexa Fluor® 488 Goat Anti-Mouse IgG H&L (ab150113, Abcam, Cambridge, United Kingdom) and incubated 1 h at 4°C. Finally, cells were washed and suspended once again in 200 mL IF media prior to analysis. For auto fluorescence measurement, cells were suspended with IF containing no Ab, whereas for isotype controls, cells were treated only with the corresponding conjugated secondary Ab. A BD LSRFortessa™ X-20v flow cytometry was used to analyze sample and at least 10,000 events were recorded for each sample. Recorded events analyzed using the FlowJo software.
4
Immunofluorescence Staining of Organoids and Intestines
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IF staining of organoids was performed as previously described19 (link). Anti-Ki67 (Abcam, 1:250), anti-GFP (Abcam, 1:1000) or FITC-anti-UEA-1 (Sigma, 1:1000) antibodies were used for staining. For IF staining of mouse intestine tissues, tissue sections were deparaffinized in xylene and rehydrated with ethanol. After pre-incubation with 2% BSA and 2% goat serum, tissue sections were incubated with the anti-LIF (Novus, 1:500), anti-lysozyme (Abcam, 1:5000), anti-Olfm4 (Cell signaling, 1:1000) or anti-GFP (Abcam, 1:1000) antibodies overnight at 4° C. Slides were then incubated with Alexa Fluor® 555 Goat Anti-Rabbit IgG (H + L) or Alexa Fluor® 488 Goat Anti-mouse IgG (H + L) (1:200). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; Vector labs).
5
Immunofluorescent Labeling of Hippocampal Tau and DAPK1
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After blocking with 5% bovine serum albumin (Proteintech) containing 1% Tririon X-100 for 1 hour at 23°C, hippocampal sections were incubated at 4°C with primary antibody overnight (DAPK1, rabbit, 1:200, CST, Cat# 3008S; Tau5, mouse, 1:200, Abcam, Cat# ab80579, RRID: AB_1603723). Then, the slices were washed and incubated with secondary antibodies – Cy3-conjugated Affinipure goat anti-rabbit IgG (1:1000, Proteintech, Cat# SA00009-2, RRID: AB_2890957) or Alexa Fluor® 488 goat anti-mouse IgG H&L (1:1000, Abcam, Cat# ab150113, RRID: AB_2576208) – for 2 hours at 23°C. The sections were then mounted on slides and sealed with a neutral resin. Images were acquired using a laser confocal microscope (FV1000 OLYMPUS, Tokyo, Japan).
6
Co-localization of IFITM1 and EphA2/EGFR
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To detect the co-localization of IFITM1 and EphA2 or IFITM1 and EGFR, cells were plated on glass-bottom cell culture plates (801006, NEST) for 24 h. After brief washing twice with 1×PBS, cells were fixed with 4% paraformaldehyde (BioSharp) and permeabilized with 0.2% Triton X-100 (Thermo Fisher, 89900). Plates were washed gently and blocked with 3% normal goat serum (C0265, Beyotime). Subsequently, the cells were incubated with the primary antibody pairs overnight, followed by incubation with respective fluorophore-conjugated secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG H&L and Alexa Fluor 647 goat anti-rabbit IgG H&L; 150113, 150079, Abcam) for 1 h and counterstained with DAPI (C1005, Beyotime) for 10 min at room temperature. To avoid false-positive cross-reactivity, primary antibody pairs were chosen from different species (mouse anti-IFITM1 and rabbit anti-EphA2, mouse anti-IFITM1 and rabbit anti-EGFR). Fluorescence images were recorded using the High Content Analysis System (CQ1, Yokogawa).
7
Immunofluorescence Staining of PANC-1 and BxPC-3 Cells
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Before adding sterile confocal glass slides (BD Biosciences) to 24-well plates, a small amount of serum-free DMEM was added to enhance the adsorption capacity of the slides. PANC-1 or BxPC-3 cells were seeded into 24-well plates and cultured for 24 h. Then, the cells were fixed with 4% paraformaldehyde on glass slides overnight at 4°C. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 for 15 min and then washed three times for 15 min. Next, 3% bovine serum albumin was used to block the cells for 30 min at 37°C. Then, the primary antibody, anti-Girdin (1:200; product code ab179481) or anti-vimentin (1:200; cat. no. 60330-1-Ig), was added to the glass slides overnight; next, the cells were washed with PBS 3 times for 15 min. The cells were then incubated with fluorescent secondary antibodies Alexa Fluor® 594 donkey anti-rabbit IgG (H+L) (1:1,000; product code ab150076) and Alexa Fluor® 488 goat anti-mouse IgG (H+L) (1:1,000; product code ab150113; both from Abcam) for ~30 min at 37°C. Finally, DAPI (blue) was used to counterstain cell nuclei for 1 min at room temperature, and the cells were examined by fluorescence microscopy (Olympus Corporation) at a magnification of ×100.
8
Immunohistochemical Analysis of Pancreatic Cells
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Pancreas were dissected and fixed overnight in 4% paraformaldehyde, washed with PBS, dehydrated, paraffin embedded, and sectioned at 5 μm. For antigen retrieval, the sections were boiled for 20 min in 10 mM of sodium citrate buffer. Sections were permeabilized with 3 × 5 min 0.1% Triton in PBS, blocked for 1 h in 5% Neutral Goat Serum (NGS) in PBS, and incubated overnight in 5% NGS with primary antibodies: mouse anti-Glucagon [K79bB10] (Abcam, ab10988), Guinea Pig anti-Insulin (Dako IR00261-2) and mouse anti-TAF4 (TAF II p135) (Santa Cruz (sc-136093). Sections were washed 3 × 5 min 0.1% Triton in PBS, and incubated with secondary antibodies: Goat anti-Guinea pig IgG H&L Alexa Fluor® 647 (Abcam ab150187) and Goat anti-Mouse IgG H&L Alexa Fluor® 488 (Abcam ab150117) for 2 h. Sections were subsequently incubated with 1/2000 Hoechst nuclear stain for 10 min, washed 3 × 5 min in PBS, dried, and mounted with Vectashild. For Taf4 and Gcg co-staining the guinea pig anti-Gcg antibody (LINCO, 4031-01 F) was used after 20 min of epitope retrieval. For GFP and Gcg co-staining, frozen cryostat sections were used without epitope retrieval. Gcg was detected with mouse anti-Glucagon [K79bB10] (Abcam ab10988) and chicken anti-mouse IgG Alexa Fluor® 647 (Thermo Scientific, # A-21463).
9
Inducing EBV Lytic Protein Expression
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EBV-positive LCL cells grown on cover slips coated with 0.1% gelatin were treated with either control (CTL) alone, 15μg/ml BA alone (BA), 3μM CDM alone (CDM), or a combination of BA and CDM (BA/CDM) for 24 hours. Cells were fixed with acetone for 10 min at room temperature. The fixed cells were then stained with anti-Zta (1:50) mouse monoclonal antibodies overnight at 4°C, and then further stained by Goat Anti-Mouse IgG H&L (Alexa Fluor® 488, obtained from Abcam #ab150113). The expression of EBV lytic proteins was visualized under fluorescence microscopy, and the nuclei of cells were stained with 40, 6-diamidino-2-phenylindole (DAPI) [36 (link), 42 (link)].
10
Immunofluorescence Analysis of Vimentin
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Cells growing on glass coverslips were processed for immunofluorescence analysis 48 h after transfection with siRNA or control. First, cells were fixed with 2% formaldehyde in PBS for 5 minutes at room temperature (RT). Following fixation, cells were washed in PBS for 10 minutes at RT. Permeabilization of the cells was achieved by incubation for 30 minutes in PBS 0.1% Triton at RT. Then blocking solution [PBS 0.1% Triton, BSA 1% and 5% normal donkey serum (NDS)] was added on top of the coverslip and incubated in a humidified chamber for 1 h. Once blocked, cells were incubated overnight at 4 °C with mouse anti-VIM ready-to-use antibody (mouse monoclonal, clone V9, catalog number IS630, DAKO, Mississauga, Canada). The next day, samples were washed 3 times with PBS during 10 minutes and incubated for 2 hours with goat anti-mouse IgG H&L (Alexa Fluor® 488, catalog number ab150113, Abcam, Cambridge, MA, USA) diluted 1:2,000 in PBS 0.1% Triton, 5% NDS. Finally, samples were washed 3 times with PBS during 5 minutes and mounted on a slide using Fluoroshield mounting medium (Sigma-Aldrich, Merck Life Science) which incorporates DAPI staining and observed under an Olympus BX51 fluorescence microscope.
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