The inhibitory capacity of borrelial proteins on the CP, LP or the AP was analysed by a microtiter-based approach as described previously59 (link). Briefly, microtiter plates were coated with either human IgM (30 ng/ml) (Merck, Darmstadt, Germany) for the CP, mannan (1 µg/ml) (Merck, Darmstadt, Germany) for the LP or LPS (100 ng/ml) (Hycult Biotech, Beutelsbach, Germany), for the AP at 4 °C overnight. Following blocking, NHS (1% for the CP, 2% for the LP, and 15% for the AP) pre-incubated with His6-tagged proteins (10 µg each or increasing concentrations thereof) were added to initiate complement activation. Formation of the MAC was detected by using an anti-C5b-9 antibody (1:500) (Quidel, Athens, USA) and antigen–antibody complexes were visualized by applying HRP-conjugated anti-mouse immunoglobulins (1:1000). The reactions were developed by adding o-phenylenediamine (Merck, Darmstadt, Germany) and measuring the absorbance at 490 nm.
Lipopolysaccharides (lps)
Manufactured by Hycult Biotech
Sourced in United States
LPS is a laboratory equipment product offered by Hycult Biotech. It serves the core function of detecting and quantifying lipopolysaccharide, a key component of the outer membrane of Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Mannan, by Merck Group (1 mentions)
Human igm, by Merck Group (1 mentions)
Anti c5b 9 antibody, by Quidel (1 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Vector unmasking solution, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Occludin, by Proteintech (1 mentions)
F4 80, by Wuhan Servicebio Technology (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Abcam (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
E selectin, by MyBioSource (1 mentions)
Cobas 6000, by Roche (1 mentions)
Kaluza acquisition software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Immunoprep reagent system, by Beckman Coulter (1 mentions)
6 protocols using lipopolysaccharides (lps)
1
Complement Inhibition by Borrelial Proteins
2
Immunohistochemical Analysis of LPS in Tissue
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Immunohistochemical (IHC) analysis of LPS was performed on formalin-fixed, paraffin-embedded tissue samples, as described [16 (link)]. Four μm-thick sections were deparaffinized, rehydrated, and rinsed. For antigen retrieval, the sections were microwaved in Vector Unmasking Solution (Vector Laboratories Burlingame, CA, USA) and treated with 3% hydrogen peroxide. Sections were incubated with LPS (Hycult Biotech, USA) monoclonal primary antibody at a 1:100 dilution. Secondary antibodies and Avidin/Biotin complex were from the Vector Vectastain ABC Elite Kit. For visualization, sections were treated with DAB (Dako Carpinteria, CA. USA), counterstained with hematoxylin, dehydrated, and mounted in a xylene-based mounting media.
Quantification was performed using open source FIJI image software using 10 images, per section, per time point, per animal. The positive signal was isolated via color threshold; percent area positive was measured and averaged.
Quantification was performed using open source FIJI image software using 10 images, per section, per time point, per animal. The positive signal was isolated via color threshold; percent area positive was measured and averaged.
3
Immunohistochemical and Immunofluorescence Analysis of Colon Tissue
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Immunohistochemical staining was performed using a goat anti-rabbit IgG (H + L) IHC kit (Servicebio, Wuhan, China). Colon slices were incubated with F4/80 (1:300, Servicebio), then stained with a DAB working solution, while the nucleoli were stained with hematoxylin. For IF staining, colon tissues were incubated with LPS (1:50, Hycult Biotech, Netherlands), ZO-1 (1:200, Servicebio, Wuhan, China), and Occludin (1:200, Proteintech, Chicago, United States) antibodies. The slices were then stained with the Alexa Fluor 594 conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:500, CST, Massachusetts, USA), and Alexa Fluor 594 conjugated goat anti-Mouse IgG (H + L) secondary antibody (1:500, CST). The nuclei were stained with DAPI.
4
Immunohistochemical Analysis of Brain Tight Junctions
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The brains were dissected, fixed in 4% paraformaldehyde for 3 h, and dehydrated in 20 and 30% sucrose for 24 h each. Frozen 6 μm sections were sliced. The primary antibodies included rabbit polyclonal ZO-1 (1:100, Invitrogen, United States), rabbit polyclonal occludin (1:100, Invitrogen, United States), and mouse polyclonal LPS (1:100, Hycult Biotech, United States). Donkey anti-rabbit IgG Alexa Fluor 488 (1:200, Abcam, United States) and donkey anti-mouse IgG Alexa Fluor 594 (1:200, Abcam, United States) were used as the corresponding secondary antibodies. DAPI (Abcam, United States) was added to the sections for counterstaining. Samples were observed at 63 × magnification under laser confocal microscope (Leica, Germany). These sections were used to analyze the mean optical intensity of ZO-1, occludin, and LPS (Ahn et al., 2018 (link); Liu et al., 2021 (link)).
5
Cardiometabolic Biomarkers in Children
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Glucose, insulin, total cholesterol, low density lipoprotein-cholesterol (LDL-C), high density lipoproteincholesterol (HDL-C), triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase were assayed on children's blood samples after overnight fast. Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR) [33] . All analyses were performed on COBAS 6000 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). E-Selectin (MyBiosource, San Diego, USA) and LPS (HycultBiotech, Plymouth Meeting, PA, USA) measurement were performed by commercial ELISA kit following the manufacturer's instructions.
6
CD38 Expression in T-Cell Subsets
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The expression of CD38 was evaluated in CD4 + and CD8 + T-cell subsets by flow cytometry in 100μL fresh anticoagulated whole blood. The cells were labeled with the following antibodies: anti-CD38-APC-Cyanine 5.5 (APC-Cy5.5, clone HIT2, Invitrogen, Frederick, MD), anti-CD4-APC-Cyanine 7 (APC-Cy7, clone OKT4, BioLegend, San Diego, CA), anti-CD8-Pacific Blue (PB, clone SK1, BioLegend, San Diego, CA), anti-CD3-Pacific Orange (PO, clone VCHT1, Invitrogen, Frederick, MD) and incubated for 20 min at room temperature in the dark. Next, the IMMUNOPREP Reagent System (Beckman Coulter, Mervue Galway, Ireland) was added to each sample using a Coulter MULTI-Q-PREP Lysing Workstation (Beckman Coulter, Miami, FL) to lyse and fixate them.
Fluorescence was measured with a Gallios™ flow cytometer (Beckman Coulter, Miami, FL). The number of events was stopped at a minimum of 200,000 cells in the lymphocyte gate for each sample and flow cytometry data were analyzed using Kaluza™ acquisition software (version 1.5; Beckman Coulter, Miami, FL). (Raybiotech, Georgia, USA), LPS (HycultBiotech, Uden, The Netherlands), LBP (R&D Systems, Minneapolis, USA) and FABP2 (Raybiotech, Georgia, USA), which were performed according to the manufacturer's procedure for each specific commercial ELISA.
Fluorescence was measured with a Gallios™ flow cytometer (Beckman Coulter, Miami, FL). The number of events was stopped at a minimum of 200,000 cells in the lymphocyte gate for each sample and flow cytometry data were analyzed using Kaluza™ acquisition software (version 1.5; Beckman Coulter, Miami, FL). (Raybiotech, Georgia, USA), LPS (HycultBiotech, Uden, The Netherlands), LBP (R&D Systems, Minneapolis, USA) and FABP2 (Raybiotech, Georgia, USA), which were performed according to the manufacturer's procedure for each specific commercial ELISA.
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