Immunofluorescent staining was performed as previously described16 (link). Following deparaffinization, antigen retrieval buffer was added to the microwaveable vessel. Liver sections were treated with PBS (containing 1% BSA) and 0.3% Triton X-100 after antigen recovery. Primary antibodies anti-MAGL (Abcam), anti-α-SMA (Abcam), anti-CD45 (Abcam) and anti-CD68 (Cell Signaling) were incubated with sections at 4 °C overnight. Sections were rinsed by PBS and secondary antibodies with Alexa Fluor 488 or Alexa Fluor 594 (Abcam) were incubated for 60 min at room temperature. VECTASHIELD antifade mounting medium with DAPI (Vector Labs) was used for DAPI counterstain and mounting.
Anti magl
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-MAGL is a lab equipment product that functions as an antibody targeting the enzyme monoacylglycerol lipase (MAGL). MAGL is responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG).
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti inos, by Proteintech (2 mentions)
Anti arg 1, by Proteintech (2 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti cd68, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Anti cd45, by Abcam (1 mentions)
Alexa fluor 594, by Abcam (1 mentions)
Fluoview fv3000, by Olympus (1 mentions)
Anti inos, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Chemidoc touch gel imaging system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti cd206, by Proteintech (1 mentions)
Aim 5 serum free medium, by Thermo Fisher Scientific (1 mentions)
Aa d8, by Cayman Chemical (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Histopaque, by Merck Group (1 mentions)
7 protocols using anti magl
1
Immunofluorescent Staining of Liver Sections
2
Synovial Tissue Immunofluorescence Staining
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The synovial tissues were permeabilized in 0.1% Triton X-100 for 20 min and then blocked with 5% bovine serum albumin for 1 h at room temperature. Then the synovial tissues were incubated with anti-MAGL (1:200, Abcam, cat. no. ab246902) and anti-iNOS (1:200; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-17139) antibodies overnight at 4°C, followed by incubation with the secondary antibody (1:1,000, Abcam, cat. nos. ab150084 and ab150113) for 2 h at room temperature in the dark. An Olympus Fluoview FV3000 (Olympus Corporation) confocal microscope was used to capture images.
3
Western Blot Analysis of Liver Proteins
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Proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer and a protease inhibitor cocktail (Sigma Life Science) from liver tissues and each concentration was measured by BCA Protein Assay Kit (Thermo scientific). Protein samples were loaded on precast gels and blotted onto Blot two Dry Blotting System (Thermo scientific). The membranes were treated with 5% skim milk and incubated with the different primary antibodies anti-MAGL (Abcam), anti α-SMA (Abcam) and anti-β-actin (Sigma Life Science) at 4 °C overnight. After TBST wash, membranes reacted with secondary antibodies. Protein bands were quantified using ChemiDoc Touch Gel Imaging System (Bio-Rad). The immunoblot band analysis for determining intensity was imaged using Image lab (Bio-Rad).
4
Synovial Tissue Immunohistochemistry Protocol
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Immunohistochemistry was performed as previously described (26 (link)). Briefly, the synovial tissues were cut into 7 µm thick sections. After blocking in 5% goat serum in PBS for 1 h at room temperature, the tissue sections were incubated with primary antibodies against anti-MAGL (1:100, Abcam, cat. no. ab246902), anti-iNOS (1:200, ProteinTech Group, Inc., cat. no. 22226-1), anti-Arg1 (1:200, ProteinTech Group, Inc., cat. no. 16001-1), anti-CD80 (1:300, ProteinTech Group, Inc., cat. no. 14292-1), or anti-CD206 (1:300, ProteinTech Group, Inc., cat. no. 18704-1) overnight at 4°C followed by the secondary antibody (1:1,000, Abcam, cat. no. ab6721) for 2 h at room temperature. An Olympus CH30 (Olympus Corporation) microscope was used to capture images (×200 or ×400).
5
Detailed Lipidomic Analysis of Primary Cells
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Histopaque, p-nitrophenyl valerate (pNPVa), and LPS (E. coli 055:B5) were purchased from Sigma (St. Louis, MO, USA). AIM V® Serum Free Medium was purchased from Thermo-Fisher (Waltham, MA, USA). RIPA lysis buffer and protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; 4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF; bestatin; pepstatin A; leupeptin hemisulfate; and aprotinin) were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for Western blots (anti-CES1, anti-MAGL, β-actin, goat anti-rabbit, and goat anti-mouse) were purchased from Abcam (Cambridge, MA, USA). Authentic 2-AG, anandamide, AA, and its deuterated analog AA-d8 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Small molecules JZL184 and WWL113 were purchased from Sigma. CpG was purchased from InvivoGen (San Diego, CA, USA). Primary CES1 and MAGL antibodies for flow cytometry were purchased from Abcam. Fluorescence secondary antibodies for flow cytometry and antibodies against IL-6 used to neutralize IL-6 or perform ELISA were purchased from Biolegend (San Diego, CA, USA). For some of the experiments, monocyte-depleted (n = 1) and whole PBMCs (n = 5) were purchased from Astarte Biologics (Bothell, WA, USA).
6
Western Blot Analysis of Synovial Proteins
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Western blotting was performed as previously described (28 (link)). The protein concentrations of the synovial tissues or cells were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (30 µg) were separated by SDS-PAGE on a 10% gel and transferred to PVDF membranes (MilliporeSigma). The PVDF membranes were blocked with 5% non-fat dry milk for 2 h, followed by incubation with one of the following primary antibodies at 4°C overnight: Anti-MAGL (1:1,000, Abcam, cat. no. ab246902), anti-iNOS (1:1,000, ProteinTech Group, Inc., cat. no. 22226-1), anti-Arg1 (1:5,000, ProteinTech Group, Inc., cat. no. 16001-1), anti-TNF-α (1:1,000, ABclonal, cat. no. A20851), anti-IL-1β (1:1,000, ABclonal, cat. no. A16288), anti-IL-6 (1:1,000, ABclonal, cat. no. A11115), anti-PTEN-induced kinase 1 (PINK1) (1:1,000, ProteinTech Group, Inc., cat. no. 23274-1), anti-Parkin (1:1,000, ProteinTech Group, Inc., cat. no. 14060-1), or anti-β-actin (1:5,000, ProteinTech Group, Inc., cat. no. 81115-1). The following day, the membranes were washed and incubated with the secondary antibody (1:5,000, Abcam, cat. no. ab205718) for 2 h at room temperature. The proteins were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher).
7
Western Blot Analysis of DAGL and MAGL
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Liver tissue samples were homogenized in a RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA) supplemented with a complete protease inhibitor cocktail (Sigma, ST. Louis, MO, USA) and centrifuged at 20,000× g for 15 min. The supernatant was collected, and protein concentration was measured with a BCA protein quantification kit (Pierce, Thermo Fisher, Waltham, MA, USA). Then, 60 µg of protein was loaded in each well and separated on 12% precast gels (Bio-Rad, Hercules, CA, USA) and blotted onto nitrocellulose membrane (Bio-Rad), which were blocked in 5% BSA and incubated with anti-DAGLβ (Cell Signaling, Danvers, MA, USA, Cat# D4P7C) dilution 1:1000 and anti-MAGL (Abcam, Cambridge, UK, Cat# ab24701) dilution 1:200. Blots were incubated with HRP-conjugated anti-rabbit secondary antibody (Abcam, ab6721) and detected using ECL solution (Cytiva, Amersham, UK). Imaging and quantification were performed with ChemiDoc (Bio-Rad).
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