104 LT73 cells were grown on Lab-Tek (ThermoFisher, Waltham, MA) slides and incubated with BSA 2% + NGS 5% blocking solution for 30 minutes, incubated with anti-human CD133/1 (Miltenyi; Biotec) for 1 hour at RT, then 30' at RT with AlexaFluor 488 goat anti human IgG (H+L) (Invitrogen) washed in tween 1× and mounted with the VECTASHIELD Mounting Medium, containing DAPI (Vector Laboratories, Burlingame, CA).
Alexa fluor 488 goat anti human igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Alexa Fluor 488 goat anti-human IgG(H+L) is a fluorescently labeled secondary antibody. It is designed to detect and bind to human immunoglobulin G (IgG) antibodies. The Alexa Fluor 488 dye provides a green fluorescent signal that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Ab33922, by Abcam (2 mentions)
Alexa fluor 594 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fv1000 confocal microscope, by Olympus (2 mentions)
Pannoramic 250 slide scanner, by 3DHISTECH (2 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 647 goat pab to rb igg, by Abcam (1 mentions)
Tcs sp2, by Leica (1 mentions)
Sars cov 2 spike chimeric monoclonal antibody, by Sino Biological (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
10 protocols using alexa fluor 488 goat anti human igg h l
1
Immunostaining of Human CD133 in LT73 Cells
2
Live Cell-Based Assay for AQP4 and MOG Autoantibodies
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HEK293T cells, polyethylenimine (PEI) transfected with human M23-AQP4, FL-MOG, or C-terminal–truncated human MOG (short-length MOG; SL-MOG) were used as the substrate for live CBAs, which were performed as described elsewhere.7 (link)– (link)9 (link) Patient sera were tested at 1:20 dilution. The Alexa Fluor 488 goat anti-human IgG (H + L) from Invitrogen (A1013, Carlsbad, CA) was used at 1:750 dilution. A semiquantitative scoring system was used: 0, no binding; 1, low-level binding; 2–4, increasing level of specific binding. Samples scoring >1 were considered positive. The average of 2 individual's scores is plotted (M.W., P.W.).
3
Visualizing HSV-2 Protein Localization
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When the 293 cells were 70%-90% confluent, they were transfected with the eukaryotic vectors of intact ICP35, VP22a and VP24 according to the Lipofectamine™ 3000 Reagent Protocol in glass bottom cell culture dishes, and the samples were harvested at 18h. In addition, Vero cells were infected with the HSV2 virus at an MOI of 0.2, and the samples were harvested at 4h, 12h, 24h postinfection. These dishes were fixed in 4% paraformaldehyde for 30 min and then blocked using 4% bovine serum albumin (BSA). For detection of the HSV-2 ICP35, VP22a, VP24 antigen and trace ICP35 protein during viral infection, the dishes were sequentially incubated with primary antibody convalescent serum, immune serum of ICP35 and secondary antibody Alexa Fluor® 647 Goat pAb to Rb IgG (Lot: GR33281 42-4, Abcam), Alexa Fluor® 488 Goat anti-human IgG (H+L) (Lot: 2196582, Invitrogen). All cell nuclei were detected with DAPI. Fluorescence was visualized and analyzed using a confocal microscope (TCS SP2, Leica).
4
Immunofluorescence Staining for GFAP and Iba1
5
Immunofluorescent Staining of GFAP and Iba1
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Slides were thawed at RT, rehydrated in PBS for 40 minutes, permeabilized in 0.1% Triton X (in PBS) for 10 minutes, washed twice for 5 minutes in PBS, and blocked overnight at 4°C in 3% BSA (in PBS). The next day, slides were incubated with rabbit polyclonal antibody against GFAP (Abcam, ab33922) at 1:5,000 or with rabbit anti–ionized calcium-binding adapter molecule 1 antibody (FUJIFILM Wako Chemicals, 019-19741) at 1:1,000, for 24 hours in 3% BSA (in PBS) at 4°C. The next day, slides were washed thrice for 5 minutes in PBS before incubation with Alexa Fluor 594 goat anti-rabbit secondary antibody (Invitrogen, Thermo Fisher Scientific, A-11012) at 1:1,000 and Alexa Fluor 488 goat anti-human IgG(H+L) at 1:500 (Invitrogen, Thermo Fisher Scientific, A-11013) in PBS for 2 hours at RT. Samples were imaged on Olympus FV1000 confocal microscope using Fluoview software. Minimal postimaging processing was done with FIJI (ImageJ, NIH).
6
Immunofluorescence Staining of Tissue Slides
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Slides were thawed at room temperature (RT), rehydrated in PBS for 40 minutes, permeabilized in 0.1% Triton X (in PBS) for 10 minutes, washed twice 5 minutes in PBS, and blocked overnight at 4°C in 3% BSA (in PBS). The next day, slides were incubated with Alexa Fluor 488 goat anti-human IgG(H+L) (Invitrogen, Thermo Fisher Scientific, A-11013) at 1:500 (in 3% BSA/PBS) for 2 hours at RT. Slides were imaged at Manchester University, Bioimaging Facility, on a 3DHistech PANNORAMIC 250 slide scanner at original magnification 20×.
7
SARS-CoV-2 Spike Protein Immunofluorescence Assay
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In longer-term SARS-CoV-2-infected and PI-treated cultures, following cell splitting and treatment, replicate cell cultures were seeded in 8-well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA). The next day, cells were fixed, and virus was inactivated by submersion into methanol for 20 min. Chamber slides were washed twice with PBS-Tween and then incubated with primary antibody, SARS-CoV-2 spike chimeric monoclonal antibody (no. 40150-D004; Sino Biological, Beijing, China), diluted 1:1,000 in PBSK for 2 h at room temperature. Following 2 washes with PBS-Tween, chamber slides were incubated with secondary antibody, Alexa-Fluor 488 goat anti-human IgG(H+L) (no. A-11013; Invitrogen, Paisley, UK), diluted 1:500 and Hoechst 33342 (Invitrogen) diluted 1:1,000 in PBS-Tween for 20 min at room temperature. The percentage of SARS-CoV-2 spike protein-positive cells was evaluated by fluorescence microscopy (Axio Vert.A1; Zeiss, Jena, Germany), assigning the following designations: 0% infected cells (no cells infected), single infected cells, and 10% to 90% infected cells (in steps of 10%). The images were acquired with ZEN 3.0 software.
8
Immunofluorescence Staining of Tissue Slides
9
Flow Cytometric Assay for Anti-GBS Antibody
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Deposition of anti-GBS antibody onto the surface of whole GBS bacteria was measured on formaldehyde-fixed GBS using a flow cytometric assay performed in 96-well microtitration plates. Briefly, 2 μL of each test serum was added to 198 μL of serotype Ia, Ib, II, III or V GBS bacteria at 5.14 × 107 CFU/mL in blocking buffer (1% BSA in PBS). This was incubated at 25 °C for 30 min with shaking (900 rpm), then pelleted. Supernatant was removed and the pellet washed twice with 200 μL of blocking buffer. Alexa Fluor® 488 Goat Anti-Human IgG (H&L) (Life Technologies) (1:500) in blocking buffer was added and samples incubated for 20 min at 4 °C, before being washed twice more with blocking buffer.
10
High-Throughput Flow Cytometry hmAb Assay
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A high-throughput multi-well plate format was used to assess reactivity of hmAbs in flow cytometry experiments. In each well, 60 μl of bacterial suspensions normalised to OD 600 of ~0.5 were mixed with 1 μg/ml of hmAb and incubated for 45 minutes at room temperature. Unbound hmAb was removed by one wash with PBS (3000 x g, 5 minutes). 5-FAM SE-stained bacteria were resuspended in 5 μg/ml of the secondary antibody, Alexa Fluor® 488 Goat Anti-Human IgG (H+L) (Life Technologies™), and the suspension incubated for 30 minutes at room temperature. Subsequently, cells were washed to remove unbound antibodies before resuspending in PBS. Fluorescence intensities were measured and analysed using a BD LSRFortessa™ Cell Analyzer (BD Biosciences) and FlowJo software version 10. Heat maps were generated using GraphPad Prism v9.4.1.
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