After overnight fixation in 4% PFA, the brains were processed for paraffin embedding, and once included, serial sagittal 4 μm thickness sections were cut using a microtome (Microm HM 340E). The slides were dewaxed using xylol and hydrated in decreasing concentration of ethanol until PBS 0.1 M, and after hydration, the immunofluorescence assay was performed as follows. Briefly, brain sections containing the hippocampus were subjected to heat-induced epitope retrieval and blocked with 10% BSA for 90 minutes at room temperature. Then, samples were immunolabeled at 4°C overnight using the following primary antibodies at 1 : 100 dilution: anti-Iba1 (GTX101495, Genetex), anti-CD68 (GTX37743, Genetex), and anti-TNFα (GTX110520, Genetex). The next day, the slides were washed and incubated with the secondary antibody (SAB4600310, Sigma Aldrich Co.) diluted at 1 : 500 during 3 hours at room temperature. Then, sections were washed repeatedly and covered with Vectashield medium (Vector Laboratories, Burlingame CA) containing DAPI to counterstain nuclei.
Anti tnfα
Manufactured by GeneTex
Sourced in United States
Anti-TNFα is a lab equipment product that serves as a tool for the detection and quantification of tumor necrosis factor alpha (TNF-α) in various research applications. It provides a reliable and efficient method to measure TNF-α levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Gtx101495, by GeneTex (1 mentions)
Gtx110520, by GeneTex (1 mentions)
Anti iba1, by GeneTex (1 mentions)
Sab4600310, by Merck Group (1 mentions)
Anti cd68, by GeneTex (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Anti il 10, by Cell Signaling Technology (1 mentions)
Anti mouse hrp, by Cell Signaling Technology (1 mentions)
Anti il 6, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Tissueruptor, by Qiagen (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Anti ly6g clone 1a8, by BioLegend (1 mentions)
4 protocols using anti tnfα
1
Immunofluorescence Analysis of Neuroinflammation
2
Protein Isolation and Quantification from Adult Heart Tissue
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Adult heart tissues were collected immediately after euthanization, and the proteins were isolated using homogenization in RIPA buffer (Sigma) with complete mini EDTA‐free protease inhibitor cocktail (Roche, Basel, Switzerland) and PhosSTOP phosphatase inhibitor (Roche) using a Qiagen TissueRuptor (Hilden, Germany). Methodologies related to protein estimation and quantitation have been described.13 The following antibodies were used: anti–extracellular signal–regulated kinase (total ERK [catalog No. 9102] and phospho‐ERK [catalog No. 9101], Cell Signaling Technology); anti–protein kinase B (total AKT [catalog No. 9272] and phospho‐AKT [catalog No. 4060], Cell Signaling Technology); anti‐JNK (total JNK [catalog No. 9252] and phospho‐JNK [catalog No. 4668], Cell Signaling Technology); anti–NF‐κB P65, (total P65 [catalog No. 8242] and phospho NF‐κB P65 [catalog No. 3033], Cell Signaling Technology); anti–IL‐6 (catalog No. 12912; Cell Signaling Technology); anti–TNF‐α ([catalog No. GTX110520], GeneTex, Irvine, CA); anti–IL‐10 (catalog No. 12163, Cell Signaling Technology); anti‐SNRK (catalog No. GTX111380, GeneTex); antirabbit HRP (horseradish peroxidase, catalog No. 7074, Cell Signaling Technology), and antimouse HRP (catalog No. 7076, Cell Signaling Technology). Quantification was done using ImageJ software and plotted against the respective controls.
3
Histological and Molecular Analyses of Lung Injury
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Primary antibodies used in the western blotting included anti-GAPDH (Santa Cruz), anti-phospho- ERK (T202/Y204, Cell Signaling Technology), anti- ERK (Santa Cruz), and anti-PCNA (Cell Signaling Technology). The haematoxylin and eosin stain (H&E) was performed by the pathology core at the Institute of Biomedical Sciences, Academia Sinica. The lung injury score was calculated according to the scoring system established by the ALI in Animals Study Group 31 (link). For immunohistochemistry staining, neutrophils were stained by anti-Ly6G (clone 1A8, Biolegend) antibody at 1:100 dilution and proliferating cells were stained by anti-Ki-67 (Cell signaling) antibody at 1:400 dilution. ImmPRESS™ HRP anti-rat IgG, mouse adsorbed (peroxidase) polymer detection kit (Vectors laboratories), or VECTASTAIN Elite ABC HRP kit (Vectors laboratories) was used for signal amplification and DAB peroxidase (HRP) substrate kit (Vectors laboratories) was used for color development. For frozen tissue staining, anti-TNF-α (Genetex) antibody was used at 1:100 dilution and Alexa Fluor 488-labeled anti-rabbit IgG (Abcam) secondary antibodies were used at 1:400 dilution. Fluoroshield mounting medium with DAPI (Abcam) was used for nuclei staining. Images were captured by LSM 700 laser scanning confocal microscope (Carl Zeiss Microscopy) and analyzed by ZEN imaging software.
4
Cardiac Injury Biomarker Evaluation
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Isoproterenol was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). ELISA kits for rat IL-6 (Cat No. MM-0190R1), rat TNF-α (Cat No. MM-0180R1), rat CK-MB (Cat No. MM-0625R1M), rat MDA (Cat No. MM-0385R1), and rat SOD (Cat No. MM-0386R1) were all purchased from Jiangsu Enzyme Free Industrial Co., Ltd. (Jiangsu, China). Anti-IL-6 antibody (Cat No. 21865-1-AP) was obtained from Proteintech Group, Inc. (Wuhan, Hubei, China). Anti-NF-κB P65 (phospho-Ser536) antibody (Cat No. YP0191) was purchased from ImmunoWay Biotechnology Company (Plano, TX, USA). Antibody for anti-TLR4 (Cat no. SAB-35463) was purchased from SAB (College Park, Maryland, USA). Antibodies for Anti-NF-κB P65 (Cat No. GTX107678) and Anti-TNF-α (Cat No. GTX35134) were obtained from GeneTex (San Antonio, Texas, USA).
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