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Hrp anti mouse

Manufactured by Agilent Technologies
Sourced in United Kingdom, United States

The HRP anti-mouse is a laboratory reagent used for the detection and quantification of mouse-derived proteins in various analytical applications. It consists of horseradish peroxidase (HRP) conjugated to an antibody that specifically binds to mouse immunoglobulins. This product can be used in techniques such as Western blotting, ELISA, and immunohistochemistry to visualize and measure the presence of mouse proteins in samples.

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6 protocols using hrp anti mouse

1

Western Blot Analysis of Cellular Proteins

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SDS-PAGE was performed using 8% and 12.5% acrylamide gels. For the Western blotting procedure, 0.2 μm nitrocellulose membranes were used. Following protein transfer to membranes, these were blocked in 5% fat-free milk powder, 5 mM NaN3 and 0.1% Tween-20 in PBS.
Antibodies and their sources were: anti-HA (Roche, 15645900), anti-Cdc48 (own production), anti-α-tubulin (Abcam, YL1/2 MA1-80017), anti-Pma1 (Abcam, 40B7 Ab4645), anti-RGS-His (Qiagen, 34610), anti-Vinculin (Sigma, hVIN1 V9264), anti-GAPDH (Cell signalling technology, 14C10 2118), anti-Na/K ATPase α-1 (Merck, C464.6 05–369), anti-Hsp105 (HSPH1, Abcam, Ab108625), anti-HSPA4 (Abcam, Ab185219), anti-Ubiquitin (Dako, Z0458), anti-Myc (Chromotek, 9E1 9e1-100), anti-Rpn1 (Enzo Life Sciences, p112-1 PW9270), anti-20S α-subunits (Enzo Life Sciences, MCP231 PW8195), anti-Hsp70 (Invitrogen, 5A5 MA3-007), anti-Aspartoacylase (Thermo Scientific, PA5-29180), anti-GFP (Chromotek, 3H9 3h9-100). Secondary antibodies and their sources were: HRP-anti-rat (Invitrogen, 31470), HRP-anti-mouse (Dako, P0260), HRP-anti-rabbit (Dako, P0448).
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2

Immunohistochemical Analysis of Myostatin

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Paraffin sections (5 μm) of 2% paraformaldehyde-fixed tissues were deparaffined, hydrated and subjected to antigen-retrieval (microwave oven treatment in 0.1 M sodium citrate). Staining was performed after quenching of endogenous peroxidase with 3% H2O2 in methanol. Slides were incubated with primary antibody overnight, followed by incubation with biotinylated antibody for 30 minutes. Each sample was analyzed for the detection of Mstn (mouse monoclonal ab, biorbyt, Cambridge, UK) and a labelled polymer HRP anti-mouse from Dako was used as secondary antibody. Staining with CD45 (Novocastra, Leica Microsystem, Milano Italy), α-SMA (Dako Italia s.r.l, Agilent Pathology Solution, Cernusco sul Naviglio, Italy) were completed with the appropriate secondary antibody using the streptavidin-peroxidase method, performed as previously described55 (link). The expression of Mstn was examined by image analysis and expressed as positive areas. In order to evaluate the co-distribution of two different antigens in the same sample, a double immunohistochemistry procedure was carried out. First, one antibody was evidenced by streptavidin-peroxidase and the second by alkaline phosphatase-conjugated streptavidin (Vector Laboratories, CA, USA). The alkaline phosphatase substrate was Vector ®RED Substrate (Vector Laboratories).
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3

Quantification of LRP-1 in Neutrophils

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Neutrophil suspensions (1 × 106/mL) were cytospun on culture glass slides positively charged. Then slides were fixed in cold methanol and staining was performed after quenching of endogenous peroxidase with 3% H2O2 in methanol. Slides were incubated with primary antibody overnight, followed by incubation with biotinylated antibody for 30 min. Each sample was analyzed for the detection of LRP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a labelled polymer HRP anti-mouse from Dako was used as secondary antibody. Staining was completed using the streptavidin-peroxidase method and DAB (3,3′-Diaminobenzidine) (Abcam, Cambridge, UK) [34 (link)]. Cytospins were counterstained with hematoxylin and mounted in Eukitt (Merck Group, Darmstadt, Germany), examined by light microscopy (Leica, Cambridge, UK), and evaluated by image analysis (Leica Q500 MC Image Analysis System, Leica). Isotype-matched IgG monoclonal antibodies (Santa Cruz Biotechnology) was tested as a negative control.
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4

Immunohistochemical Analysis of Tissue Samples

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Tissue specimens were fixed in neutral formalin buffered saline (10%)
and embedded in paraffin. Hematoxylin and eosin staining was performed using
standard methods and tissue specimens from experimental animals were reviewed in
a blinded fashion by a clinical pathologist (M. M. I.) For Ki67 staining,
3–4 micron tissue sections were cut from paraffin blocks and baked
overnight in a dry slide incubator, then deparaffinized on a Shandon-Lipshaw
Varistain using a series of incubations in xylene, ethanol, then water. Antigen
retrieval was achieved by incubating slides in Tris-HCL 9.0 AR buffer in a T-FAL
OPTIMA pressure cooker. Slides were rinsed, then endogenous peroxidase was
blocked by immersing slides in 3% hydrogen peroxide for 5 minutes. Following
washes in nanopure water and TBS-20, primary antibody was applied at a dilution
of 1:200 (Ki67, MIB-1 Clone, Dako). Following primary antibody incubation,
slides were washed and then incubated with envision-labelled polymer-HRP
Anti-Mouse (Dako). Slides were washed, then DAB+ solution (DakoCytomation) was
added for a 15-minute incubation, after which slides were rinsed with nanopure
water. Chromogen signal was enhanced using DAB Sparkle Enhancer (Biocare).
Slides were washed, then counterstained with Harris Hematoxylin, dehydrated,
cleared, then mounted using Cytoseal (VWR).
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5

Immunohistochemical Analysis of Tissue Samples

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Tissue specimens were fixed in neutral formalin buffered saline (10%)
and embedded in paraffin. Hematoxylin and eosin staining was performed using
standard methods and tissue specimens from experimental animals were reviewed in
a blinded fashion by a clinical pathologist (M. M. I.) For Ki67 staining,
3–4 micron tissue sections were cut from paraffin blocks and baked
overnight in a dry slide incubator, then deparaffinized on a Shandon-Lipshaw
Varistain using a series of incubations in xylene, ethanol, then water. Antigen
retrieval was achieved by incubating slides in Tris-HCL 9.0 AR buffer in a T-FAL
OPTIMA pressure cooker. Slides were rinsed, then endogenous peroxidase was
blocked by immersing slides in 3% hydrogen peroxide for 5 minutes. Following
washes in nanopure water and TBS-20, primary antibody was applied at a dilution
of 1:200 (Ki67, MIB-1 Clone, Dako). Following primary antibody incubation,
slides were washed and then incubated with envision-labelled polymer-HRP
Anti-Mouse (Dako). Slides were washed, then DAB+ solution (DakoCytomation) was
added for a 15-minute incubation, after which slides were rinsed with nanopure
water. Chromogen signal was enhanced using DAB Sparkle Enhancer (Biocare).
Slides were washed, then counterstained with Harris Hematoxylin, dehydrated,
cleared, then mounted using Cytoseal (VWR).
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6

Protein Extraction and Western Blot

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Cells were harvested and lysed in 1x ice-cold Cell Lysis Buffer (Cell Signaling Technology) supplemented with 1x Complete Protease Inhibitor Cocktail–EDTA free (Roche) and 1 mM PMSF. Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (BioRad), and probed for overnight with primary antibodies at 4°C. The membrane was incubated with secondary antibodies including: conjugated horseradish peroxidase (HRP) anti-mouse (DAKO, 1:5000) and HRP anti-rabbit (DAKO, 1:2000). The membrane was exposed to X-ray film (Fujifilm) and the film was developed in automatic X-ray film processor.
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