Glass slides were coated with amine solution as described in a previous report25 (link). Evaluation of the acquired immune response was assessed in 32 pairs of vivax sera samples (D0, D28, and 1 yr). Sera pooled from eight vivax malaria patients or eight knowlesi malaria patients were used to evaluate cross-immunoreactivity. The crude rPvAARP-FL and rPkAARP-FL proteins were spotted in duplicate, incubated at 37 °C for 2 h, blocked with 5% BSA in PBS 0.1% Tween-20, and incubated at 37 °C for 1 h. The rPvAARP-FL-coated slides were reacted with sera from malaria patients or healthy individuals (1:25 dilution) at 37 °C for 1 h. Alexa Fluor 546-conjugated goat anti-human IgG (10 µg/mL; Invitrogen Life Technologies) was used as a secondary antibody, and the fluorescence signal was detected with a scanner (InnoScan scanner, Carbonne, France). The cut-off values were set to the mean fluorescence intensity (MFI) plus 2 standard deviations (SDs). The normalized MFI was calculated by dividing the MFI by the cut-off value.
Alexa fluor 546 conjugated goat anti human igg
Manufactured by Thermo Fisher Scientific
Sourced in France
Alexa Fluor 546-conjugated goat anti-human IgG is a fluorescent-labeled secondary antibody. It is designed to detect and label human immunoglobulin G (IgG) in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Innoscan scanner, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fluorescence scanner, by PerkinElmer (1 mentions)
Alexa fluor 546 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti rat igg h l, by Thermo Fisher Scientific (1 mentions)
Herceptin, by Roche (1 mentions)
Perjeta, by Roche (1 mentions)
Dfc3000 g, by Leica (1 mentions)
5 protocols using alexa fluor 546 conjugated goat anti human igg
1
Serological Evaluation of Vivax and Knowlesi Malaria
2
Quantitative Serological Profiling of P. knowlesi
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Amine-coated slides were used and spotted in duplicate with 100 ng of purified recombinant proteins of PkDBPα or PkAMA1 at 37 °C for 2 h. After blocking with 5% BSA in PBS 0.1% Tween-20, knowlesi-infected patients’ serum and healthy individuals’ serum were reacted (1:25) for 1 h. Alexa Fluor 546-conjugated goat anti-human IgG (10 µg/mL; Invitrogen Life Technologies) was used as a secondary antibody, and the fluorescence signal was detected with a scanner (InnoScan scanner, Carbonne, France). The cut-off values were set to the mean fluorescence intensity (MFI) plus 2 standard deviations (SDs). The normalized MFI was calculated by dividing the MFI by the cut-off value.
3
Evaluating PvETRAMPs Antibody Reactivity
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The preparation of amine-coated slides was described in a previous report [25 (link)]. Serum samples from P. vivax malaria patients or healthy individuals were used to evaluate the antibody reactivity of PvETRAMPs. A recombinant protein (100 ng in each protein) was spotted in each well of a slide, followed by incubation for 2 h at 37 °C. After washing the slide with phosphate-buffered saline-Tween (PBST, 0.1%), the slide was blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. The slide was probed with serum (1:10 dilution) and incubated for 1 h at 37 °C. Alexa Fluor 546-conjugated goat anti-human IgG (10 ng/μl; Invitrogen, Carlsbad, CA, USA) was spotted on the slide, followed by incubation for 1 h at 37 °C for visualization. The slide was scanned in a fluorescence scanner (Perkin Elmer, Boston, MA, USA) [34 (link)]. The mean fluorescence intensity (MFI) of each spot was quantified using ScanArray Express software version 4.0 (Perkin Elmer). The cut-off MFI value was determined as the MFI of serum from a healthy individual plus two standard deviations [33 (link)].
4
Multiparametric Imaging of Cell Surface Receptors
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Trastuzumab (Herceptin®, Roche, binds HER2); pertuzumab (Perjeta®, Roche, binds HER2); TS2/16.2.1 mouse monoclonal produced in-house from hybridoma (ATCC Cat# HB-243, binds integrin β1); BIIG2 rat monoclonal produced in-house from hybridoma (DSHB Antibody Registry ID: AB_528155, binds α5 integrin); ab528 mouse monoclonal produced in-house from hybridoma (ATCC Cat# HB-8509 binds: epidermal growth factor receptor EGFR);Alexa Fluor 546 conjugated goat anti-Mouse IgG (H + L), highly cross-adsorbed (ThermoFisher cat# A-11030); Alexa Fluor 647 conjugated Goat anti-Mouse IgG (H + L) highly cross-adsorbed (ThermoFisher cat# A-21235); Alexa Fluor 546 conjugated Goat anti-Human IgG (H + L) highly cross-adsorbed (ThermoFisher cat# A-21089); Alexa Fluor 647 conjugated Goat anti-Human IgG (H + L) highly cross-adsorbed (ThermoFisher cat# A-21445);Alexa Fluor 647 conjugated Goat anti-Rat IgG (H + L) highly cross-adsorbed (ThermoFisher cat# A-21247).
5
Quantifying MDA5 Protein Expression
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The CBAs were performed as previously described [13 (link)]. HEK293T cells were co-transfected with MDA5 and pcDNA3.1-EGFP using Lipofectamine 2000 (Thermo, #11668027). EGFP works as an internal control to show transfected cells that express MDA5 protein. Merged IF can help to show that a positive signal is specifically distributed in transfected cells. The cells were fixed with 4% paraformaldehyde (PFA), blocked in 10% goat serum, and permeabilized with 0.2% Triton X-100. Alexa Fluor 546-conjugated goat anti-human IgG (Thermo, #A-21089) was used at a 1:500 dilution. The fluorescent signal was observed and imaged under a Leica DFC3000G. Serum was diluted at 1:10 in the original test. Two independent masked assessors classified each sample as positive or negative based on the intensity of IF in direct comparison with that of non-transfected cells and control samples. Once confirmed, the positive specimens were then serially diluted at 1:10, 1:30, 1:100 and 1:300 to determine the titres. The final titre was defined as the sample dilution value for which specific fluorescence was barely but clearly identifiable and expressed as the corresponding dilution value.
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