S. schenckii and S. brasiliensis were analyzed for their interaction with hMDM in the presence of wHS or heat-inactivated human serum (iHS) as well as to evaluate phagocytic index upon blocking CR3, TLRs and Dectin-1. To block CR3, TLR2, TLR4, and Dectin-1 receptors, monoclonal anti-CD11b (Thermo Fisher, MA5-16528), anti-TLR2 (Abcam, ab16894), anti-TLR4 (Abcam, ab22048), and anti-Dectin-1 antibodies (35 (link)) were used, respectively. Briefly, hMDMs were preincubated with fresh DMEM medium supplemented with 10% of wHS for 30 min at 37°C in an atmosphere of 5% of CO2. Monoclonal antibodies were then added in a final concentration of 10 µg/ml and incubated further at 8°C for 1 h. Then, hMDMs were infected with FITC-labeled yeasts of S. schenckii or S. brasiliensis, and the interaction was allowed for a duration of 1 h at 37°C in a CO2 incubator. The hMDMs were washed one time with PBS (GIBCO®, NY, USA) pH 7.4 and incubated for 15 min at ambient temperature with Trypan blue (GIBCO®, NY, USA) at a final concentration of 1.2 mg/ml. The cells were washed twice with PBS and fixed with 1% p-formaldehyde for 15 min at ambient temperature. Finally, hMDMs were washed once with PBS and, with the aid of a cell scraper, were gently released from the wells, suspended in PBS supplemented with 3% fetal bovine serum, and analyzed in a BD FACS-Canto™ II.
Ab16894
Manufactured by Abcam
Sourced in United Kingdom
Ab16894 is a laboratory reagent product manufactured by Abcam. It is a primary antibody used for research purposes. The core function of this product is to detect and bind to a specific target protein or antigen in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab22048, by Abcam (4 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Af534, by R&D Systems (1 mentions)
Ab103791, by Abcam (1 mentions)
Ab150064, by Abcam (1 mentions)
Ab66705, by Abcam (1 mentions)
Ab213676, by Abcam (1 mentions)
Ab150109, by Abcam (1 mentions)
Ab64482, by Abcam (1 mentions)
Ab15104, by Abcam (1 mentions)
Goat anti rabbit igg hrp sc 2004, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab9722, by Abcam (1 mentions)
Ab1793, by Abcam (1 mentions)
Phosphatase inhibitor, by Solarbio (1 mentions)
Ab133739, by Abcam (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
6 protocols using ab16894
1
Blocking Phagocytosis Receptors in Sporothrix Interactions
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S. schenckii and S. brasiliensis were analyzed for their interaction with hMDM in the presence of wHS or heat-inactivated human serum (iHS) as well as to evaluate phagocytic index upon blocking CR3, TLRs and Dectin-1. To block CR3, TLR2, TLR4, and Dectin-1 receptors, monoclonal anti-CD11b (Thermo Fisher, MA5-16528), anti-TLR2 (Abcam, ab16894), anti-TLR4 (Abcam, ab22048), and anti-Dectin-1 antibodies (35 (link)) were used, respectively. Briefly, hMDMs were preincubated with fresh DMEM medium supplemented with 10% of wHS for 30 min at 37°C in an atmosphere of 5% of CO2. Monoclonal antibodies were then added in a final concentration of 10 µg/ml and incubated further at 8°C for 1 h. Then, hMDMs were infected with FITC-labeled yeasts of S. schenckii or S. brasiliensis, and the interaction was allowed for a duration of 1 h at 37°C in a CO2 incubator. The hMDMs were washed one time with PBS (GIBCO®, NY, USA) pH 7.4 and incubated for 15 min at ambient temperature with Trypan blue (GIBCO®, NY, USA) at a final concentration of 1.2 mg/ml. The cells were washed twice with PBS and fixed with 1% p-formaldehyde for 15 min at ambient temperature. Finally, hMDMs were washed once with PBS and, with the aid of a cell scraper, were gently released from the wells, suspended in PBS supplemented with 3% fetal bovine serum, and analyzed in a BD FACS-Canto™ II.
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S. schenckii and S. brasiliensis were analyzed for their interaction with hMDM in the presence of wHS or heat-inactivated human serum (iHS) as well as to evaluate phagocytic index upon blocking CR3, TLRs and Dectin-1. To block CR3, TLR2, TLR4, and Dectin-1 receptors, monoclonal anti-CD11b (Thermo Fisher, MA5-16528), anti-TLR2 (Abcam, ab16894), anti-TLR4 (Abcam, ab22048), and anti-Dectin-1 antibodies (35 (link)) were used, respectively. Briefly, hMDMs were preincubated with fresh DMEM medium supplemented with 10% of wHS for 30 min at 37°C in an atmosphere of 5% of CO2. Monoclonal antibodies were then added in a final concentration of 10 µg/ml and incubated further at 8°C for 1 h. Then, hMDMs were infected with FITC-labeled yeasts of S. schenckii or S. brasiliensis, and the interaction was allowed for a duration of 1 h at 37°C in a CO2 incubator. The hMDMs were washed one time with PBS (GIBCO®, NY, USA) pH 7.4 and incubated for 15 min at ambient temperature with Trypan blue (GIBCO®, NY, USA) at a final concentration of 1.2 mg/ml. The cells were washed twice with PBS and fixed with 1% p-formaldehyde for 15 min at ambient temperature. Finally, hMDMs were washed once with PBS and, with the aid of a cell scraper, were gently released from the wells, suspended in PBS supplemented with 3% fetal bovine serum, and analyzed in a BD FACS-Canto™ II.
2
Immunofluorescence Analysis of TLR2, Serpin E1, PLAUR, BNP, OSM, PGP9.5, NeuN, and PLAUR
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Paraffin sections were de‐paraffinized, rehydrated, permeabilized, followed by incubation with rabbit antibody to TLR2 (1:300, Abcam ab213676), Serpin E1 (1:300, Abcam ab66705), PLAUR (1:300, Abcam ab103791), BNP (1:100, Abcam ab236101), OSM (1:75, Thermo PA576861), mouse antibody to PGP9.5 (1:300, Abcam ab8189), NeuN (1:500, Novus NBP1‐92693), TLR2 (1:300, Abcam ab16894), or Antigen Affinity‐purified polyclonal goat IgG against PLAUR (10ug/ml, AF534, R&D) in blocking solution (4°C, overnight). The samples were washed in PBS and incubated with donkey anti‐rabbit Alexa 594 (1:500, Abcam ab150064) or anti‐mouse Alexa 488 (1:500, Abcam ab150109). Subsequent to a final wash, specimens were mounted onto slides using prolonged anti‐fade reagents containing DAPI (ThermoFisher Scientific) and captured using an IX73 Olympus microscope. Fluorescence intensity was analyzed using CellSens Dimension Imaging software and Image J.
3
Quantification of mRNA and Protein Levels
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mRNA and protein determination was performed using q-PCR and western blot (WB) assay as described in detail previously [26 (link)]. For real-time PCR, all primer sequences were designed by NCBI GenBank and produced by Sangon Biotechnology Ltd. (Shanghai, China) (S1 Table and S2 Table ). For western blot analysis, primary antibodies against NOD2 (rabbit monoclonal antibody [EPR16252], ab197030), TNFα (mouse monoclonal antibody [52B83], ab1793), IL-1β (rabbit polyclonal antibody, ab9722), TLR2 (mouse monoclonal antibody [T2.5], ab16894), TLR4 (mouse monoclonal antibody [76B357.1], ab22048), Occludin (rabbit polyclonal antibody, ab64482) and claudin4 (rabbit polyclonal antibody, ab15104) were purchased from Abcam (UK). Secondary antibodies of goat anti-mouse IgG-HRP (sc-2005) and goat anti-rabbit (sc-2004) IgG-HRP were purchased from Santa Cruz (USA). The targeted proteins were and visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo scientific pierce) and the intensity of visualized bands were analyzed by using Quantity One software (Bio-rad). β-actin was used as an internal control. Data were expressed by the ratio to β-actin.
4
Zymosan-Induced Protein Analysis in OSCC Cells
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OSCC cells treated with zymosan (100 μg/mL) were collected and lysed on ice in RIPA buffer with phosphatase inhibitor (Solarbio, Beijing, China) and phenylmethanesulfonylfluoride (PMSF). After centrifugation, the protein concentration in supernatants was determined by an Enhanced BCA Protein Assay Kit (Beyotime, Haimen, China), and the protein samples were then incubated at 100 °C for 10 min. Equal amounts of total protein were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% nonfat milk powder in Tris-buffered saline/Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibody overnight at 4 °C (TLR2, ab16894, Abcam; MyD88, ab133739, Abcam; E-cadherin, ab40772, Abcam; NF-κB p65, #8242, Cell Signaling Technology; p-NF-κB, #3033, Cell Signaling Technology and β-actin, A1978, SIGMA) and then incubated with HRP-conjugated secondary antibodies (anti-rabbit IgG from Sigma-Aldrich and anti-mouse IgG from Cell Signaling Technology) at a dilution of 1:5000 for 1 h at room temperature. Protein bands were visualized using High-sig ECL substrate and a Tanon 5200 CE machine (Tanon, Shanghai, China).
5
Immunofluorescence Analysis of Hippocampal TLR Pathway
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After dewaxing, the wax pieces of hippocampal tissue were immersed in a 3% hydrogen peroxide solution for 15 min, washed with PBS three times, and subjected to antigen retrieval by a 0.1 M sodium citrate solution. It was blocked in goat serum and incubated at 37°C for 30 min. Then, the wax pieces of hippocampal tissue were incubated with primary antibodies (anti-TLR2, Abcam, ab16894; anti-TLR4, Abcam, ab22048; anti-MyD88, Abcam, ab28763; and anti-NF-κB, Abcam, ab16502; Cambridge, UK) overnight at 4°C, washed with PBS three times, incubated with the appropriate secondary antibodies labeled with fluorescence for 30 min at 37°C, and then washed with PBS three times. Finally, the sections were stained with DAPI and incubated for 10 min at room temperature and then washed with PBS three times, sealed with a neutral resin, and observed under a fluorescence microscope.
6
Protein Extraction and Western Blot Analysis
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Primary cells were lysed with RIPA buffer and a proteinase inhibitor mixture (PMSF). Nuclear and cytoplasmic proteins were separated using a nuclear and cytoplasm protein extraction kit (78,833, Thermo Pierce, Rockford, IL, USA), according to the manufacturer’s instructions. The protein concentration was assessed using a Multiskan instrument (Thermo, Multiskan Mk3). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (Immobilon-P PVDF Membrane, Merck Millipore). Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 h at room temperature and incubated overnight with primary anti-α-SMA (ab32575, Abcam) (1:5000), anti-vimentin (ab92547, Abcam) (1:5000), anti-TLR2 (ab16894, Abcam) (1:1000) and anti-TLR4 (ab22048, Abcam) (1:1000) at 4 °C. After washing, an HRP-conjugated secondary antibody was added for 1 h at 37 °C. Detection was performed with enhanced chemiluminescence (ECL), and blots were quantified by densitometry using the accompanying computerized image analysis program (Image Tool).
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