Cells were grown on coverslips overnight. Then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS for 15 min in room temperature. Then aspirate fixative, rinse three times in 1 × PBS for 5 min each. After treating with 0.5% Triton-X100 (ThermoFisher, USA) diluted in warm PBS and blocking specimen in blocking buffer (5% Bovine Serum Albumin diluted in warm PBS, BioFroxx, Guangzhou, China) for 60 min in room temperature, primary antibodies were applied for 4 C overnight. AlexaFluor488- conjugated anti-mouse IgG (1:1000, Molecular Probes, A11029), AlexaFluor488-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11034) or AlexaFluor594-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11037) secondary antibodies were used in room-temperature for 1 h. Nuclei were counterstained with DAPI (1 μg/ml, Molecular Probes, D1306). Protein subcellular localization was analyzed under a Zeiss 510 META or Leica TCS-SP2 confocal laser scanning microscope. The concentration of primary antibody was rabbit anti-CD68 (1:200, Abcam, ab125212), rabbit anti-CD163 (1:200, Proteintech, 16,646–1-AP), rabbit anti-CD204 (1:200, Abcam, ab123946), rabbit anti-CD206 (1:200, Abcam, ab64693), mouse anti-Arg1 (1:200, Abcam, ab239731), rabbit anti-Mrc1 (1:200, Abcam, ab64693), rabbit anti-Fizz1 (1:200, Abcam, ab39626).
Ab39626
Manufactured by Abcam
Sourced in United States
Ab39626 is a lab equipment product. It serves as a core function without further details on intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab15323, by Abcam (2 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
Ab123946, by Abcam (1 mentions)
Ab239731, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
510 meta, by Leica (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tcs sp2, by Leica (1 mentions)
A21203, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluoview 1000, by Olympus (1 mentions)
Image pro plus software, by Adobe (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Alexa fluor 488 594 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Sc 377009, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
7 protocols using ab39626
1
Immunofluorescence Analysis of Macrophage Markers
2
Histological and Immunofluorescence Analysis of Epididymal Adipose Tissue
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Epididymal fat was harvested at indicated time points (six mice per group for each time point). The epididymal adipose tissues were fixed in formalin and embedded with paraffin. Sections were cut at 6 μm and stained with hematoxylin-eosin (HE) according to a standard protocol. The size of adipocytes was measured using digital image analysis. For immunofluorescence, the adipose tissues were fixed in 4% paraformaldehyde, dehydrated with 50% sucrose, and embedded in OCT (Sakura, Finetek, USA, 4583). Frozen sections (10 μm thick) (3 slides per mouse) were blocked in BSA and incubated overnight at 4 °C with primary antibodies against Fizz1 (Abcam, San Francisco, USA, ab39626) or iNOS (Abcam, ab15323), followed by incubation with Alexa Fluor 488/594-conjugated secondary antibodies (Invitrogen, A21203, A31556, A-32766) at room temperature for 2 h. Finally, 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, B2261) was added. Sections were observed using a confocal laser scanning microscope (Olympus FluoView 1000, Tokyo, Japan, TCSSP8). BMDMs were plated onto glass coverslips and fixed with 4% paraformaldehyde. The next steps were performed as described above.
3
Quantitative Analysis of FIZZ1 Protein Expression
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Immunohistochemistry has been applied for determining where the FIZZ1 protein was localized and its concentration. Lung paraffin sections were deparaffinized, rehydrated, and blocked by 3% H2O2. Sections were then blocked with goat serum at a concentration of 5% to reduce the absorption of non-specific immunoglobulins. After that, the specimens were incubated with anti-FIZZ1 antibody (1:1,000, ab39626, Abcam) at 37°C for 2 h, followed by incubating with a secondary antibody at 37°C for 30 min. Image-Pro Plus software (Version X; Adobe, San Jose, CA) has been applied for evaluating protein expression. In total, eight images of bronchioles from each tissue section were examined using ×400 magnification and the program Case Viewer (version 1.3; 3DHistech, Budapest, Hungary). Data were presented as an average optical density (AOD) for the entire sections of lung tissue showing positive staining.
4
Comprehensive Immunohistochemical Analysis of Mouse EAT
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Mice were anaesthetized with intraperitoneal injection of sodium pentobarbital and then underwent consecutive perfusions of 20 ml of PBS and 20 ml of 4% paraformaldehyde through the right ventricle. For H&E staining, EAT was embedded in paraffin, sectioned at 10 μm thickness and then stained with H&E according to the standard protocol. For immunofluorescence staining, EAT was dehydrated through incubation in 30% sucrose/PB overnight, embedded in optimal cutting temperature compound (OCT, Sakura, Finetek, USA) and cut into frozen sections at 10 μm. The frozen sections were blocked with BSA and then incubated at 4 °C overnight with indicated primary antibodies against Fizz1 (ab39626, Abcam, San Francisco, USA), iNOS (ab15323, Abcam, San Francisco, USA) and F4/80 (sc-377009, Santa Cruz, USA), followed by further incubation with Alexa Fluor 488/594-conjugated secondary antibodies (1: 500, Life Technologies), shielding from light, at room temperature for 2 h. Finally, the sections were incubated with 4′-6-diamidino-2-phenylindole (DAPI) for 7 min for nuclear staining and imaged with a laser scanning confocal microscope (Leica, Wetzlar, Germany).
5
Quantification of Lung Protein Profiles
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Pulverized mouse lungs were homogenized in RIPA buffer (50 mM Tris HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA) supplemented with an EDTA-free protease inhibitor (Roche) and protein extracts were quantified. Western blotting was performed using 15% SDS-polyacrylamide gels and membranes were probed with antibodies raised against β -actin (Santa Cruz, sc1616, 1:1000) and RELMα (Abcam, ab39626, 1:1000). Blots were incubated with horseradish peroxidase-linked secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific) and visualized with a digital luminescent image analyzer (Image Quant LAS4000 Mini; GE).
6
Immunohistochemical Analysis of Lung Tissue
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Lung tissue was embedded in paraffin after overnight fixation in 4% paraformaldehyde. Microwave-induced antigen retrieval (15′ at 240 W) was performed in 0.01 M sodium citrate (pH 6). For immunohistochemistry analysis, endogenous peroxidase was blocked with 3% H2O2 in methanol. Antibody binding was visualized using a LSAB + Peroxidase Kit with 3,3′ -diaminobenzidine as the chromogen (Dako). Sections were dehydrated and mounted with Eukitt mounting medium (Sigma-Aldrich). For immunohistochemistry and immunofluorescence, sections were stained overnight at 4 °C with antibodies against Ki67 (Abcam, ab16667,1:300 for IHC and 1:100 for IF), RELMα (Abcam, ab39626, 1:300 for IHC and 1:50 for IF), HIF2α (Abcam, ab199, 1:50), CC10 (Santa Cruz, sc9772, 1:50) and acetyl-tubulin (Sigma-Aldrich, clone 6-11B-1, 1:3000). For immunodetection of BrdU positive cells, the antibody (BD Biosciences, 347580, 1:50) was incubated for 1 h at room temperature. The percentage of Ki67 and BrdU-positive cells in each experimental condition was calculated by counting bronchial epithelial cells in different bronchial airways of each mouse.
7
Lung Tissue Protein Expression Analysis
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The proteins from lung tissues have been extracted utilizing the RIPA Lysis Buffer containing protease inhibitor (GRF101, Epizyme) and phosphatase inhibitor cocktails (GRF102, Epizyme) while quantification performed utilizing a BCA Protein Assay Kit (ZJ101, Epizyme). Equivalent amounts of proteins have been electrophoresed on polyacrylamide gels and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated for 2 h at room temperature by the primary antibodies which included ß-Tubulin antibody (HX 1829, huaxingbio), anti-FIZZ1 antibody (1:1,000, ab39626, Abcam) p38 mitogen-activated protein kinase (MAPK) Rabbit mAb (#8690, Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) Rabbit mAb (#4511, Cell Signaling Technology). Following a 40-min incubation at room temperature with secondary antibodies, the membranes were detected using a chemiluminescence substrate system (Bio-Rad Laboratories, CA, United States). The band intensities of proteins have been quantitated using Image J software.
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