Lung slices (with recovered antigens) were reacted with primary antibodies (mouse anti-rat ED1 antibody for total macrophage [Millipore MAB1435]; rabbit anti- rat iNOS antibody for M1 macrophage [Abcam ab15323]; mouse anti- rat CD163 antibody for M2 macrophage [BioRad MCA342R]; rabbit anti- human Podoplanin gp36 antibody for human alveolar type I cell [Abcam 236529]; mouse anti-toll-like receptor-4 antibody [TLR-4, Abcam ab30667, specific for rat and human]; mouse anti-α-smooth muscle actin antibody for myofibroblast [SMA, Sigma A2547]) at 4°C for 12- 18 h and then reacted with secondary antibodies. Samples were then reacted with avidin-biotinylated-horseradish peroxidase complex (ABC Kit, Vector Laboratories) and finally developed with DAB. Finally, the numbers of M1 macrophage or M2 macrophage were counted from ten optic fields in three left lung sections of each group.
Ab30667
Manufactured by Abcam
Sourced in United States
Ab30667 is a laboratory product. It is a polyclonal antibody that targets a specific protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab76003, by Abcam (2 mentions)
Mouse anti β actin antibody, by Merck Group (2 mentions)
Test tube, by BD (2 mentions)
Ab12085, by Abcam (2 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Mab1435, by Merck Group (1 mentions)
Mca342r, by Bio-Rad (1 mentions)
Ab15323, by Abcam (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Bay 11 7082, by Beyotime (1 mentions)
Ab72100, by Abcam (1 mentions)
Fluorescein fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence ecl detection system, by Keygen Biotech (1 mentions)
P8340, by Merck Group (1 mentions)
P5726, by Merck Group (1 mentions)
Ab104836, by Abcam (1 mentions)
Ab2185, by Abcam (1 mentions)
Ab137852, by Abcam (1 mentions)
10 protocols using ab30667
1
Lung Slice Immunohistochemistry Assay
2
Molecular Signaling Pathways in LPS-Induced Inflammation
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LPS was purchased from Sigma (St. Louis, MO, USA). Primary antibodies against CXCR4 (40kDa, ab80791), CXCR7 (42kDa, ab72100) and TLR4 (92kDa, ab30667) were purchased from Abcam Ltd. (Cambridge, UK). Rabbit anti-human antibodies against p38 MAPK (43kDa), phospho-p38 MAPK (43kDa), ERK1/2 (42/44kDa), phospho-ERK1/2 (42/44kDa), JNK (54kDa), phosphor-JNK (54kDa), NF-κB p65 (65kDa), phosphor-IKKɑ/β (85/87kDa), MEK1/2 inhibitor U0126 and JNK inhibitor SP600125 were purchased from Cell Signal Technology (Beverly, MA, USA). Rabbit polyclonal IκBɑ (41kDa), IKKɑ/β (85/87kDa) and phosphor-IκBɑ (41kDa) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated monoclonal mouse anti-GAPDH antibody was purchased from Zen BioScience (Chengdu, China). Fluorescein (FITC)-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The NF-κB inhibitor BAY 11–7082 was obtained from Beyotime Institute of Biotechnology (Shanghai, China). The BCA protein assay kit and enhanced chemiluminescence (ECL) detection system were purchased from KeyGEN (Nanjing, China).
3
Protein Extraction and Western Blot Analysis
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The radioimmunoprecipitation assay (RIPA) buffer was used to extract total protein, supplemented with 1% protease inhibitors (P8340, Sigma-Aldrich) and phosphatase inhibitors (P5726, Sigma-Aldrich). The bicinchoninic acid (BCA) assay was used to measure protein concentration. Western blot analysis was performed, as previously described (12 (link)). THBS2 (sc-136238, Santa Cruz Biotechnology), TLR4 (ab30667, Abcam), Ki67 (Proteintech Group, Inc.), HIF-1α (ab2185, Abcam), GLUT1 (ab115730, Abcam), HK2 (ab104836, Abcam), ALDOA (ab150396, Abcam), PKM2 (ab137852, Abcam), and LDHA (ab101562, Abcam) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H+L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) were obtained from Proteintech Group, Inc. (Jackson).
4
Quantification of Lung Tissue Proteins
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Lung tissue samples from each group were loaded separately into individual wells. The polyvinylidene fluoride (PVDF) paper was reacted with primary antibodies (mouse anti-α-SMA antibody, rabbit anti-MMP-9 antibody [Abcam ab76003], mouse anti-Toll-like receptor-4 antibody [TLR-4, Abcam ab30667], and mouse anti-β-actin antibody [Sigma A5411] for internal control) at 4 °C for 12–18 h. Subsequently, the membrane was reacted with the corresponding secondary antibodies at room temperature for 1 h. The protein bands were quantified using ImageJ software and normalized using individual internal controls for comparison.
5
Protein Quantification in Lung Tissue
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Lung tissue samples from each group were loaded separately into individual wells. The polyvinylidene fluoride (PVDF) paper was reacted with primary antibodies (mouse anti-α-SMA antibody, rabbit anti-matrix metallopeptidase 2 antibody [MMP-2, Abcam ab92536, specific for rat and human], rabbit anti- MMP-9 antibody [Abcam ab76003, specific for rat and human], mouse anti-toll-like receptor-4 antibody [TLR-4, Abcam ab30667, specific for rat and human], and mouse anti-β-actin antibody [Sigma A5411] for internal control) at 4 °C for 12- 18 h. Subsequently, the membrane was reacted with the corresponding secondary antibodies at room temperature for 1 h. The protein bands were quantified using Image J software and normalized using individual internal controls for comparison.
6
Characterization of hTMSC Surface Antigens
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The specific surface antigens of hTMSCs were characterized by flow cytometry analysis. The cells were incubated into a test tube (BD, Franklin Lakes, NJ) at a density of 1×105 cells/ml, then washed three times with wash buffer (PBS and 3% FBS). The cells were incubated with primary antibody for 40 min with saturating concentrations of monoclonal antibodies CD14 (all anti-human CD from BD Biosciences, San Jose, CA), CD19, CD29, CD34, CD73, CD90, HLA-DR, TLR2 (all anti-human TLR from Abcam, Cambridge, ab9100), TLR3 (ab12085), TLR4 (ab30667), and TLR5 (ab13875). After the cells were washed three times in buffer and centrifuged at 1200 rpm for five minutes, they were resuspended in ice cold PBS and incubated with the secondary antibody for 30 min in the dark at 40°C. Cell fluorescence was evaluated by flow cytometry in a FACS Caliber instrument (BD) and the data were analyzed using Cell Quest software (BD).
7
Quantification of Cell Surface Markers in hTMSCs
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For the measurements of cell surface markers via flow cytometry, the hTMSCs were plated at a density of 1 × 105 cells/ml into a test tube (BD, Franklin Lakes, NJ) and washed three times with wash buffer (PBS with 3% FBS) as previously described [12 (link)]. The antibodies against CD14 (all anti-human CD from BD Biosciences, San Jose, CA), CD19, CD34, CD73, CD90, CD105, HLA-DR, TLR 2 (ab9100) (all anti-human TLR from Abcam, Cambridge), TLR 3 (ab12085), TLR 4 (ab30667), and TLR 5 (ab13875) were added to the incubation of the hTMSCs as the primary antibody. Cell fluorescence was evaluated by flow cytometry using a FACS-Calibur instrument (BD).
8
Western Blot Analysis of Lung Proteins
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Total proteins were harvested from the lung tissue. Equal amounts of proteins (50 µg/lane) were separated by 10% polyacrylamide gel electrophoresis, and the proteins were transferred electrophoretically onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then incubated with primary antibodies. The primary antibodies and their concentrations were as follows: anti-TLR4 (1:1,000 dilution; ab30667), anti-caspase-3 (1:1,000 dilution; ab52293) and anti-β-actin (1:5,000 dilution; ab8226) (all from Abcam). After being washed with TBST 3 times, the membranes were then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and anti-mouse IgG-HRP; Cat. nos. sc-2004 and sc-2005, respectively; 1:2,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
9
Antibody Profiling for Vascular and Inflammatory Signaling
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The reagents used in study were anti-vascular endothelial growth factor (VEGF) antibody (ab46154; Abcam, Cambridge, UK), von Willebrand factor (vWF) antibody (11778-1-AP; Proteintech Group Inc., Rosemont, IL, USA), anti-toll-like receptor (TLR)-4 antibody (ab30667; Abcam), anti-nuclear factor kappa B (NF-κB) p65 antibody (ab16502; Abcam), anti-interleukin (IL)-17A (ab134086; Abcam), anti-IL-17 rabbit pAb (WL02981; Shenyang Wanlai Biotechnology, Shenyang, Liaoning Province, China) and the Cell Counting Kit (E1CK-000208; EnoGene, Nanjing, Jiangsu Province, China) (Table 1 ).
10
Optimized Peptide Characterization and Antibody Analysis
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The materials and chemical characteristics of the optimized peptides used in this study were as follows: the peptides were kindly provided from Nutrily Biotechnology, Ltd. (Anyang, Henan, China) and prepared as previously outlined.24,26 (link) The antibodies were as follows: CD-68 (ab125212; Abcam, Cambridge, MA, USA), MPO (ab9535; Abcam, Cambridge, MA, USA), CD31 (ab182981; Abcam, Cambridge, MA, USA), normal goat serum (ab7481; Abcam, Cambridge, MA, USA), TLR4 (ab30667; Abcam, Cambridge, MA, USA), phospho-Ser536 NF-κB/p65 (#3033; Cell Signaling Technology, Danvers, MA, USA), NF-κB/p65 (#8242; Cell Signaling Technology, Danvers, MA, USA) and β-actin (#12620; Cell Signaling Technology, Danvers, MA, USA).
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