After MRI, lesions were excised, embedded in paraffin, and cut into 5 μm sections. Sections were deparaffinized in xylene, rehydrated, and antigen retrieval was performed with 10mM sodium citrate buffer (pH 6) for 20 min. Sections were quenched, blocked, and incubated overnight with primary antibodies against myelin basic protein (MBP, Dako A0623, 1:500), CD68 (microglia/macrophages; CellSignaling #76437, 1:500), iNOS (inducible nitric oxide synthase; Novus NB120‐15203, 1:300), Ferritin (abcam ab75972, 1:100), CD206 (abcam ab117644, 1:200), and MerTK (tyrosine‐protein kinase MER; abcam ab52968, 1:500). Tissue was processed with the appropriate biotinylated secondary antibodies and avidin/biotin staining kit with diaminobenzidine (DAB) as chromogen (Vector Laboratories ABC Elite Kit and DAB Kit). Negative controls included isotype‐controls and absence of immunolabeling in tissues that do not express the above antigens. DAB‐enhanced Perls’ Prussian blue was used to detect ferric iron. Slides were immersed in 4% ferrocyanide/4% hydrochloric acid for 30 min in the dark, and staining was enhanced through incubation with DAB for 30 min at room temperature. After staining, all sections were rinsed, dehydrated, cover‐slipped, and digitized using a Mirax digital slide scanner.
Ab52968
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab52968 is a laboratory equipment product manufactured by Abcam. It is a device used for scientific research and analysis purposes. No further details can be provided in a concise, unbiased, and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Mab8912, by R&D Systems (3 mentions)
Ab75972, by Abcam (1 mentions)
Ab117644, by Abcam (1 mentions)
A0623, by Agilent Technologies (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Ab109231, by Abcam (1 mentions)
Pk 6101, by Vector Laboratories (1 mentions)
Dmr microscope, by Leica (1 mentions)
Anti mertk, by R&D Systems (1 mentions)
Recombinant protein g sepharose beads, by Thermo Fisher Scientific (1 mentions)
P mertk, by PhosphoSolutions (1 mentions)
Sc 480, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Af154, by R&D Systems (1 mentions)
Complete mini, by Roche (1 mentions)
Ab169545, by Abcam (1 mentions)
Mayer s hematoxylin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions)
Universal quick kit, by Vector Laboratories (1 mentions)
13 protocols using ab52968
1
Immunohistochemical Analysis of Lesion Pathology
2
Immunostaining of Axl, Mer, and Tyro3 in OA Synovial Tissue
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Immunostaining was performed as previously described [16 (link)]. OA synovial tissues were used to determine protein expression of Axl, Mer, and Tyro3. Sections of synovial tissues were incubated with rabbit anti-human Axl (1:600; C89E7; Cell Signaling, Danvers, MA, USA), rabbit anti-human Mer (1:2000; ab52968; Abcam, Cambridge, UK), rabbit anti-human Tyro3 (1:500; ab109231; Abcam), or rabbit anti-human IgG (1:74,000; X0936; Agilent Technologies, Santa Clara, CA, USA) overnight at 4 °C and subsequently with biotinylated goat anti-rabbit IgG (1:400; PK-6101; Vector Laboratories, Peterborough, UK) for 30 min at RT. A biotin–streptavidin horseradish peroxidase detection system was used according to the manufacturer’s protocol (PK6101; Vector Laboratories). Bound complexes were visualized with diaminobenzidine by incubation for 10 min at RT. Sections were counterstained with hematoxylin. Pictures were taken with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) at 20× and 40× magnification.
3
Stabilization of Phosphorylated FLT3 and MERTK
4
Immunoprecipitation and Immunoblotting of RTKs
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Cultured cells were treated with Foretinib or vehicle for 1 h. Freshly made pervanadate (0.12 mM Na3VO4 in 0.0002% H2O2) was added to samples where indicated as a phosphate stabilizer for 5 min before collecting protein lysate. Lysis buffer (50 mM HEPES, pH 7.5, 150 nM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 0.1 mM Na2MoO4) with protease inhibitor (Complete Mini, Roche, Mannheim, Germany) was added to each well, rocked on ice for 15 min, and then scraped. The lysate was divided and incubated with the appropriate antibody (MerTK: MAB8912, R&D Systems, Minneapolis, MN; Axl: R&D Systems AF154; Tyro3: Epitomics EPR4308) and Protein G-sepharose beads 4B with rotisserie rotation overnight. Immune complexes were washed thoroughly and heated to 95 °C for 5 min in Laemmli Sample Buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 5% β-Mercaptoethanol, 2% SDS and 0.01% bromophenol blue). Samples were resolved using SDS-PAGE and transferred to nitrocellulose using the iBlot Dry Blotting System (Invitrogen, Carlsbad, CA). Nitrocellulose membranes were blotted with anti-MerTK (ab52968, Abcam, Cambridge, MA) or anti-Tyro3 (Epitomics EPR4308) or anti-Axl (R&D Systems AF154).
5
Immunohistochemical Evaluation of MerTK and Endoglin
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PDX tumors were obtained from Patient-Derived Xenograft Core, Baylor College of Medicine, and Jackson Laboratory (Bar Harbor, ME, USA). Human TNBC and normal/benign human breast tissues were obtained from Translational Research Initiatives in Pathology (TRIP)lab, UWCCC. PDX tumors, normal/benign human breast tissues, and the TNBC cancer patient TMA were stained using the Universal Quick Kit (#PK-8800, Vector Laboratories, Newark, CA, USA) according to manufacturer’s recommendation. Sections were heated in TRIS-EDTA buffer (pH 9.0) in a decloaking chamber and incubated overnight at 4 °C with MerTK (1:50, ab52968, Abcam, Cambridge, UK) or endoglin (1:250, ab169545, Abcam) antibodies or with no antibody as a control. Antibody binding was detected using 3,3′-diaminobenzidine substrates (Vector Laboratories) and counterstained with Mayer’s hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA). Samples were examined using an Olympus BX51 microscope. Images are shown at a magnification of 10×, 20×, or 40×.
6
Immunofluorescent Staining of hMERTK
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Samples were cut into 10 μm sections using a cryostat (Leica Microsystems, Wetzlar, Germany). Thereafter, sections were rinsed 3 times with PBS for 5 min at room temperature, and then blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. Sections were subsequently transferred to a moist chamber containing rabbit recombinant monoclonal anti-hMERTK antibody (ab52968; 1:200; Abcam) at 4°C overnight. Thereafter, sections were rinsed 3 times with PBS and incubated with Alexa Fluor 594 anti-rabbit secondary antibody (R37119; 1:1000; Invitrogen) in the dark for 1 h at room temperature. Cell nuclei were then stained with DAPI and fluorescent signals were visualized using a Zeiss fluorescence microscope (Observer Z1).
7
Immunohistochemical Analysis of Ocular Markers
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Antibodies used in this study are: mouse anti-pan-flavivirus (clone 4G2, MAB10216 Millipore), mouse anti-N-Cadherin (clone 5D5, Abcam), rabbit anti-ZO1 (#617300 Invitrogen), rabbit anti-β-catenin, (clone E247, Abcam), rabbit anti-MERTK antibody (#ab52968, Abcam), mouse anti-CRALBP, (#ab15051, Abcam), rabbit anti-GAPDH antibody (#G9545, Sigma-Aldrich), rabbit anti-Tyrosinase (#CSB-PA025394LA01HU, Cusabio Technology LTD), rabbit anti-PDI (#ab3672, Abcam).
8
Phosphorylated MERTK Receptor Analysis
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9
MERTK Protein Expression in hiPSC and hiPSC-RPE
10
Immunoblot Analysis of Protein Expression
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Cells were lysed on ice in a lysis buffer composed of 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) and 5 mM EDTA with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 500g for 30 min at 4°C to remove cellular debris. Proteins (25 μg/lane) were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). For the detection of α-syn, membranes were fixed in 4% paraformaldehyde solution as previously described.38 (link) Membranes were immunoblotted for AXL (AF154 R&D Systems at 1:500), MerTK (ab52968, Abcam at 1:500), phosphorylated MerTK (p186-749, Phosphosolutions at 1:1000), α-syn (ab138501, Abcam 1:5000), S129-phosphorylated α-syn (ab51253, Abcam 1:1000), CSF1R (MAB3291, R&D systems; 1:250) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G8795, Sigma Aldrich at 1:5000) overnight at 4°C, and then with horse radish peroxidase-linked secondary antibodies (1:10 000; Jackson Laboratory) for 1 h. Bands were detected by enhanced chemiluminescence with Clarity Max ECL substrates (Bio-Rad Laboratories) using a ChemiDoc Imaging System (Bio-Rad Laboratories). Image analysis was performed using ImageLab 6.0.1 software (Bio-Rad Laboratories).
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