After deparaffinization, tissue sections of RA and OA patients were pre-treated with citrate buffer (10 mM sodium citrate, pH 6.0). Endogenous peroxidase activity was disrupted with 3% H2O2. Nonspecific protein binding was blocked with 1% bovine serum albumin/ 5% goat serum for 40 minutes. Mouse monoclonal anti-p97 antibodies (Abcam), mouse monoclonal anti-HDAC6 antibodies (Santa Cruz) or mouse IgG2a (isotype control) were applied over night at 4°C. Slides were washed in PBS-T (0.05% Tween 20 in PBS) and incubated with biotinylated goat anti-mouse antibodies (1:1000; Jackson ImmunoResearch). The signal was amplified with ABC reagent (Vector Laboratories) and detected with 3,3′-diaminobenzidine (Vector Laboratories).
Biotinylated goat anti mouse antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Biotinylated goat anti-mouse antibodies are secondary antibodies that are conjugated with biotin. They are designed to bind to mouse primary antibodies, enabling their detection and visualization in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Abc reagent, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Streptavidin alexa568, by Thermo Fisher Scientific (1 mentions)
Amersham cell proliferation labeling reagent, by GE Healthcare (1 mentions)
Avidin biotin blocking, by Vector Laboratories (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Abc standard kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
4 protocols using biotinylated goat anti mouse antibody
1
Immunohistochemical Analysis of p97 and HDAC6 in RA and OA
2
Visualizing Nucleolar Dynamics with UBF
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Imaging of labelled UBF is widely used to identify undercondensed active or inactive rDNA genes folded into the structure of interphase FC and metaphase NORs [15 , 60 (link)–62 (link)]. To perform UBF labeling, AMD treated H2B-GFP transfected cells were immediately fixed at the end of time-lapse imaging. To label UBF we used: (i) mouse anti-UBF/F9-fragment monoclonal antibodies (Santa Cruz Biotechnology, USA); (ii) biotinylated goat anti-mouse antibodies (Jackson, USA); (iii) streptavidin-Alexa568 (Invitrogen Molecular Probes, USA). After immunolabeling, COI previously imaged during time-lapse experiments were found by their position on the finder grid. After 3D imaging of immunostained UBF, cells were prepared for TEM and CLEM analysis (for details see S7 Method ).
3
BrdU Incorporation Assay for Proliferation
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BrdU (5-Bromo-2-deoxyridine) incorporation assay was used to quantitate cell cycle entry and proliferation. 10 μl of BrdU (Amersham Cell Proliferation Labeling Reagent, GE Healthcare Life Sciences, Little Chalfont, UK) per gram body weight was administered via IP on alternate days following UUO. Double immunostaining was performed for BrdU and tdTomato. Antibody paraffin embedded tissue was prepared and blocked as described above. Avidin/biotin blocking (Vector Laboratories, Burlingame, CA, USA) was performed to block endogenous biotin and prevent unspecific staining while using biotin-streptavidin labeling system. Tissue was incubated overnight at 4 °C with a primary mouse anti-BrdU (1:200, Amersham, GE Life Sciences, Buckinghamshire, UK). This was followed with a biotinylated goat anti-mouse antibody (1:500, Jackson Immunoresearch Laboratories, Inc, West Grove, PA, USA) incubated at room temperature for 1 h. The signal was amplified by incubation with streptavidin-conjugated with Alexa Fluor 488 (1:100; Invitrogen, Grand Island, NY, USA) for 45 min. Negative control staining was performed by omitting primary antibody staining.
4
Quantifying FSHR Expression in Human Myometrium
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Sections of paraffin-embedded human myometrium were deparaffinized in xylene and rehydrated in an ethanol series. Immunolocalization of FSHR in human myometrium was performed as previously described [13 (link)]. FSHR-323 IgG2a and nonimmune IgG2a were each used at 5 μg/ml and were applied in blocking buffer overnight at 4°C. After samples were washed, biotinylated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) was added at 42 μg/ml for 1 h at room temperature. The ABC Standard Kit (Vector Laboratories, Inc.) was used according to manufacturer's instructions, and immunoreactivity was visualized with 3,3-diaminobenzidine (DAB; Dako North America, Inc.). Samples were counterstained with 10% Harris hematoxylin (Leica Microsystems Inc.). Images were captured using a BX61 model light microscope (Olympus). Staining was quantified using Image J software (U.S. National Institutes of Health; http://imagej.nih.gov/ij .) with the following modifications. The analysis was limited to FSHR from myometrial smooth muscle by focusing on rectangles of uniform size at the top, right, bottom, left, and center of each image, shifting these as necessary to avoid blood vessels. For each rectangle, the percentage of pixels exceeding a threshold value of 190 was determined, and then the percentages for all rectangles on a given slide were averaged.
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