Protein was isolated using a single detergent lysis buffer as reported 12 , 30 , and protein concentration was determined using the bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA). DMEM was conditioned by cells for 24 h as previously reported 19 . Equal concentration of protein or volume of conditioned medium were evaluated by Western blotting as previously described 30 using anti‐mouse SPARC antibody (AF942, R&D Systems, Minneapolis, MN, USA).
Af942
Manufactured by R&D Systems
Sourced in United States
AF942 is a recombinant human Activin A/BMP-1 Precursor protein. It is a member of the transforming growth factor beta (TGF-beta) superfamily. Activin A is involved in the regulation of cell growth, differentiation, and other functions.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements ar 4, by Nikon (2 mentions)
Dxm1200c, by Nikon (2 mentions)
Mca341r, by Bio-Rad (2 mentions)
Imagepro 6.0 plus, by Media Cybernetics (2 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Digital sight ds u3, by Nikon (1 mentions)
M7249, by Agilent Technologies (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Af3075, by R&D Systems (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Ab756p, by Merck Group (1 mentions)
Axiocam mr3, by Zeiss (1 mentions)
Ab23750, by Abcam (1 mentions)
Eclipse e800 microscope, by Nikon (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Sc 7273, by Santa Cruz Biotechnology (1 mentions)
Gtx74034, by GeneTex (1 mentions)
9 protocols using af942
1
Protein Isolation and SPARC Detection
2
Immunohistochemical Staining of Tissue Sections
3
Immunohistochemistry of Brain Tissue
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Mice were perfused by intracardial perfusion with 4% paraformaldehyde in 1× PBS. Brains were cut into 20 μm sections with a cryostat. Slides were rinsed three times in PBS for 10 min each, then incubated with blocking buffer (1% BSA, 5% normal goat or donkey serum, and 0.2% Triton X-100 in PBS) for 1 h at room temperature (RT). Primary antibodies against Ki-67 (1:100 rabbit anti-Ki-67, Pierce #PA5-19462), Sparc (1:500 goat anti-Sparc, R&D Systems #AF942), or Sox9 (1:100 goat anti-Sox9, R&D Systems #AF3075) were incubated overnight at 4°C in the appropriate blocking buffer. After washing slides three times in PBS, secondary antibody (1:2000, donkey anti-goat Alexa Fluor 647 or goat anti-rabbit Alexa Fluor 488, Life Technology) was added for 2 h at RT. For EdU immunostaining, slides were permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT. The slides were then washed with 3% BSA in PBS twice for 5 min each. The reaction cocktail was added for 30 min at RT. The sections were rinsed once in BSA for 5 min before mounting.
4
Immunohistochemical Analysis of ECM Proteins
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The following antibodies were used in this study: collagen I (600-401-103-0.1; Rockland, Gilbertsville, PA, USA), collagen IV (AB756P; EMD Millipore, Billerica, MA, USA), collagen VI (SAB4500387; Sigma-Aldrich), fibronectin (ab23750; Abcam, Cambridge, MA, USA), laminin (AB2034; EMD Millipore), and SPARC (AF942; R&D Systems, Minneapolis, MN, USA). Sections were washed with xylene and subsequently hydrated with ethanol dilutions (100%, 95%, 70%) three times. Sections were then rinsed with deionized H2O. Tissue was then blocked in 10% donkey serum (all other antibodies) for 1 hour at RT and permeabilized using 0.2% Triton-100. Primary antibody was applied at 1:100 concentration overnight at 4°C. Slides were subsequently washed with 1× Phosphate Buffered Saline with Tween20 (PBS-T), and 1:200 secondary antibody was applied for 1 hour at RT (Table 1 ). Slides were washed once again, and the tissue was imaged using the Zeiss Axiovert 200M inverted fluorescent microscope (Carl Zeiss, Heidelberg, Germany) attached to a digital camera (AxioCam MR3; Carl Zeiss). Images were obtained using the associated software (Axiovision 4.8.2; Carl Zeiss). Slides of tissue from control and experimental interventions were imaged at the same time using the same control slide to normalize the intensity of staining.
5
Histological and Immunohistochemical Tissue Analyses
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Adjacent serial sections were routinely stained with hematoxylin and eosin (H&E), immunohistochemically stained for SPARC (1:200 for 45 minutes; AF942, R&D Systems), cell proliferation marker Ki‐67 (1:250 for 40 minutes; M7249, Agilent Technologies) and macrophage/histiocyte marker CD68 (1:200 for 30 minutes; MCA341R, BioRad, Los Angeles, CA, USA) as previously reported 36 . Additional adjacent sections were subjected to the periodic acid Schiff (PAS) reaction with or without diastase (liver tissue was positive control) as previously described 43 to demonstrate phagocytic activity (histiocytes). Images of stained sections were captured on either a Nikon Eclipse E800 microscope with a Nikon DXM1200C digital camera using ImagePro 6.0 Plus (Media Cybernetics, Rockville, MD, USA) software or a Nikon Eclipse Ni microscope with a Digital Sight DS‐U3 digital camera using NIS‐Elements AR 4.20 (Nikon, New York, NY, USA) software. Composite figures were prepared using Adobe Photoshop CS6 software.
6
Collecting SPARC-Depleted Conditioned Media
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Conditioned media (CM) were collected from 4T1‐1F8 cells cultured for 72 hr in DMEM + 1% FBS. DMEM + 1% FBS served as control. CM were centrifuged, filtered (0.2 μm) and stored at −80°C until use. In some cases, CM were depleted of tumor‐cell secreted SPARC using goat anti‐SPARC (R&D AF942) coupled to protein‐G dynabeads (Thermo) by O/N incubation at 4°C under constant rotation.
7
Protein Extraction and Western Blot Analysis
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Tissues were harvested followed by snap freezing in liquid nitrogen, then homogenized in RIPA buffer with protease inhibitors. Cell lysates were prepared by harvesting in RIPA buffer with protease inhibitors. Equal amounts of protein were run on SDS-PAGE gels after quantification of protein concentration using the DC protein assay (Bio-Rad) and transferred to nitrocellulose membrane. Primary antibodies and appropriate secondary antibodies were used to probe blots. The bands were detected by chemiluminescent visualization (PI32209; Pierce). The following primary antibodies were used for experiments. Antibodies to SPARC (1:1,000; AF942, R&D), β-actin (1:1,000; 4967L, Cell Signaling), p-AKT (1:1,000; 9271S, Cell Signaling), AKT (1:1,000; 9272S, Cell Signaling), PPARγ (1:200; sc-7273, Santa Cruz), p-HSL (S563) (1:1,000; 4139, Cell Signaling), HSL (1:1,000; 4107, Cell Signaling), IL-1β (1:1,000; GTX74034, Genetex), p-JNK (1:1,000; 4668S, Cell Signaling), JNK (1:1,000; 9252S, Cell Signaling), p-p38 MAPK (1:1,000; 4511S, Cell Signaling), p38 MAPK (1:1,000; 8690S, Cell Signaling), NF-kB p65 (1:1,000; 8242S, Cell Signaling), p-NF-kB p65 (1:1,000; 3033S, Cell Signaling), STAT1 (1:1,000; 9172S, Cell Signaling), and Caspase-1 (1:250; a gift from Genentech) were used. ImageJ was used for densitometry analysis.
8
Quantifying Secreted Protein Levels
9
Histological Analysis of Tissue Samples
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Tissues were fixed in 10% neutral buffered formalin for 24 h and transferred to 70% ethanol. Tissues were embedded in paraffin and 4 μm sections were processed for H&E staining, picrosirius staining, immunohistochemistry (IHC) and co‐immunofluorescence (Co‐IF) using standard protocol as previously stated.18 The following antibodies were used: SPARC (R&D Systems, AF942, 1:100, Research Resource Identifiers (RRID): Antibody (AB)_2286625), α‐SMA (Dako, Clone 1A4, 1:250, RRID: AB_2335694), CC3 (Cell signalling, 9664L, 1:100, RRID: AB_2335694) CCL2 (Invitrogen, #MA5‐17040, Clone 2D8, 1:500, RRID: AB_2538512), CD45 (BD Biosciences, 550539, 1:100, RRID: AB_2174426).
Pictures were taken with 40x magnification for H&E and IHC staining and 20x magnification for Co‐IF staining. Olympus DP27 and Olympus confocal FV1000 cameras were used for visualization.
Pictures were taken with 40x magnification for H&E and IHC staining and 20x magnification for Co‐IF staining. Olympus DP27 and Olympus confocal FV1000 cameras were used for visualization.
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