Cells were fixed in ice-cold methanol for 20 min at −20 °C, rehydrated three times with phosphate-buffered saline (PBS), and blocked in 5% bovine serum albumin–PBS for 30 min. The cells were stained with primary antibody in blocking buffer for 1 h at 37 °C, rinsed in PBS, and stained again with Alexa Fluor 488-labeled rabbit anti-mouse IgG or Alexa Fluor 594-labeled goat anti-rabbit IgG (Life Technologies) for 1 h at 37 °C. The cells were then rinsed with PBS containing 4′, 6-diamidino-2-phenylindole (DAPI) and mounted. The cells were observed under a confocal microscope (LSM 710 NLO and DuoScan System, Zeiss).
Alexa fluor 488 labeled rabbit anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-labeled rabbit anti-mouse IgG is a fluorescently-labeled secondary antibody that specifically binds to mouse immunoglobulin G (IgG) antibodies. The Alexa Fluor 488 dye molecule is attached to the antibody, enabling fluorescent detection and visualization of mouse IgG target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Duoscan system, by Zeiss (1 mentions)
Lsm 710 nlo, by Zeiss (1 mentions)
Anti cox 4, by Abcam (1 mentions)
Alexa fluor 594 labeled donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti mterf3, by Abcam (1 mentions)
Nucblue dapi, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Synergy htx plate reader, by Agilent Technologies (1 mentions)
Bhk 21, by American Type Culture Collection (1 mentions)
Prism 7, by GraphPad (1 mentions)
4 protocols using alexa fluor 488 labeled rabbit anti mouse igg
1
Immunofluorescent Staining of Cells
2
Immunofluorescence Labeling of MTERF3 and COX IV
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Immunofluorescence experiments were performed with standard protocols using the following antibodies to label MTERF3 and COX IV: anti-MTERF3 (1:200; Abcam), and anti-COX IV (1:200; Abcam). Secondary antibodies used were Alexa Fluor 594-labeled donkey anti-rabbit IgG (1:300; Life Technologies, Carlsbad, USA) and Alexa Fluor 488-labeled rabbit anti-mouse IgG (1:2500; Life Technologies).
3
Visualization of Endocytic Trafficking
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After iBECs were pulsed with Alexa Fluor 647-labeled IgG (human or mouse, 667 nM) or human transferrin (66.7 and 667 nM, Jackson ImmunoResearch) diluted in Phenol-free Ham’s F-12 (Caisson) for 1 hour, the fluorogenic solutions were replaced with Phenol-free Ham’s F-12. Immediately after, 16% (v/v) paraformaldehyde (Electron Microscopy Sciences) was added to the medium for a final concentration of 4% (v/v). Following 30 minutes of fixation, cells were extensively rinsed with PBS. Cells were then permeabilized with 0.1% (v/v) Triton X-100 (MilliporeSigma) for 5 minutes, treated with 3 µg/mL mouse anti-LAMP2 antibody (Invitrogen) overnight at 4 °C, and then extensively rinsed with PBS. For detection, the cells were then treated with 5 µg/mL Alexa Fluor 488-labeled rabbit anti-mouse IgG (Invitrogen) for 1 hour at room temperature. Nuclei were labeled with NucBlue (DAPI; Life Technologies) as recommended by the manufacturer.
4
YF Virus Neutralizing Antibodies Determination
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YF virus-specific neutralizing antibodies were determined in baby hamster kidney cells (BHK-21, ATCC), essentially as described by.43 (link) In brief, triplicates of three-fold dilution series (starting at a dilution of 1∶50) of individual or pooled plasma samples were mixed with 20–40 TCID50 of YF 17D vaccine virus in minimum essential medium (MEM) (2.5% FCS, 1% glutamine, 0.5% neomycin) and incubated for 1 h at 37 °C prior to addition of cells and further incubation for 72 h at 37 °C. The cells were then fixed with 4% paraformaldehyde and virus-infected cells were detected using a YF virus-specific mouse monoclonal antibody (1 µg/ml 2D12; affinity-purified from ATCC CRL-1689 hybridoma cells) in combination with an Alexa Fluor 488 labeled rabbit anti-mouse IgG (Invitrogen, catalog # A11059, dilution 1:500). Fluorescence was measured in a Synergy HTX plate reader (BioTek). Neutralization titers were determined at a cut-off of 50% reduction of the fluorescence signal (NT50 titers), using a four-parameter logistic regression for curve fitting (GraphPad Prism 7; GraphPad Software Inc.).
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