Cells were lyzed in radioimmunoprecipitation assay buffer and lysates were assayed by western blot analysis for the expression of S100A8 (Abcam, Cambridge, UK, ab92331), S100A9 (Abcam, ab105472), MMP2 (Abcam, ab92536) and MMP9 (Abcam, ab137867), ERK1/2 (Cell Signaling, #9126S) or phosphorylated ERK1/2 (Cell Signaling, #4695S) using the indicated antibodies.
Ab105472
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab105472 is a lab equipment product manufactured by Abcam. It serves as a core function for laboratory applications, but a detailed description while maintaining an unbiased and factual approach cannot be provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab137867, by Cell Signaling Technology (2 mentions)
Ab92536, by Abcam (2 mentions)
Ab92331, by Abcam (2 mentions)
Ab133737, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
F4 80, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Ab93357, by Abcam (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Antibody to β actin, by Cell Signaling Technology (1 mentions)
Ab150157, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
S 1000, by Vector Laboratories (1 mentions)
A11037, by Abcam (1 mentions)
Alexa fluor 594 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
H1500, by Olympus (1 mentions)
5 protocols using ab105472
1
Protein Expression Analysis by Western Blot
2
Histopathological Analysis of Mouse Lung Tissues
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Five-micrometer sections of mouse lung tissues and other organs were placed on coated slides for haematoxylin-eosin (H&E) and immunohistochemistry staining. Each lung tissue section contained the maximum coronal planes of all five lung lobs in individual mice. For immunohistochemistry, after deparaffinization and rehydration, the sections were incubated with 3% H2O2 in distilled water to neutralize endogenous peroxidase. Antigen unmasking was performed using heat treatment with citrate solution. Then the sections were incubated overnight at 4 °C with the primary antibodies. The antibodies and dilutions used were: TTF1 (ab133737, abcam), H3K27me3 (ab6002, abcam), p-ERK (4376, Cell Signaling Technology), p-Akt (4060, Cell Signaling Technology), F4/80 (76437, Cell Signaling Technology), S100A9 (ab105472, abcam). The secondary antibodies were added and then incubated at room temperature for 1 hour before DAB reaction. Tumor numbers were counted on one H&E slide representing a complete cross section of the lungs per animal. Tumor burden was shown as the percentage of total tumor area over total lung area. Tumor area and total lung area quantification were performed by ImageJ measurements, and 10 randomly chosen fields from the lung lobes of each mouse were measured 23 (link).
3
Protein Expression Analysis of Innate Immune Markers
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The joint and lung tissue lysates (20 μg per sample) were resolved on NuPage 4–12% Bis-Tris protein gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were blocked overnight with 5% milk powder (w/v) and probed with antibodies for murine CRAMP (rabbit polyclonal, Abcam, USA, catalog number ab93357), S100A8 (rat monoclonal [clone ABM4A69], Abcam, catalog number ab220174), S100A9 (rat monoclonal [clone 2B10], Abcam, catalog number ab105472), α-defensin 1 (goat polyclonal, Abcam, catalog number ab122884), β-defenisn 2 (rabbit polyclonal, MyBioSource, USA, catalog number MBS2005685) and β-defensin 14 (rabbit polyclonal, MyBioSource, catalog number MBS1490249). Antibody to β-actin (Cell Signaling Technologies) was used to normalize for protein loading. Affinity-purified horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling, USA) along with Amersham ECL Prime (GE Healthcare) was used for detection. The blots were imaged using AmershamTM Imager 680 blot and gel imager. Densitometry assessment of band intensity was determined using AmershamTM Imager 680 analysis software version 2.0. The relative band intensity was assessed after normalization with the band intensity for β-actin.
4
Quantification of Neutrophil Infiltration in Dorsal Root Ganglia
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Mice were anesthetized by overdose with pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde in 1X PBS. DRGs were dissected and fixed in 4% PFA for 2 h, followed by 30% sucrose with 0.02% azide in 1X PBS. DRGs were placed in OCT and cut on a cryostat at 10 μm, followed by mounting on SuperFrost Plus glass slides for staining. Infiltration of neutrophils into the DRG neurons in naïve, control, and primed mice was evaluated using immunohistochemical staining of S100A9 costained with NeuN, a marker for neurons. The tissue sections were blocked with 10% normal goat serum (Vector Labs S-1000) in PBSTx (0.3% Triton-X in 1×PBS). Sections were costained with a monoclonal rat anti-S100A9 (Abcam; ab105472) and recombinant rabbit anti-NeuN antibody (Abcam; ab177487) and their respective secondary antibodies (Alexa Fluor 594 anti-rabbit IgG, ThermoFisher; A11037 and Alexa Fluor 488 goat anti-rat, Abcam; ab150157). Sections were mounted and coverslipped with Vectashield Hardset Antifade Mounting Medium with DAPI (Vectorlabs; H-1500) and imaged using an Olympus fluorescent microscope. The total number of S100A9+ cell neurons was quantified in three DRG sections per slide and pooled per mouse using ImageJ. Neutrophils within 250 μm of the tissue periphery were counted by a blinded experimenter using ImageJ.
5
Western Blot Analysis of Inflammation Markers
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