Animals for Experiments 1 and 3 were perfused transcardially with buffered saline (100 ml), followed by buffered 4% paraformaldehyde in 0.2 M PBS (500 ml). For animals that underwent cortical lesioning, the brain and spinal cord were then removed, post-fixed in the same buffered 4% paraformaldehyde for 24 hr and finally cryoprotected in 30% sucrose until the tissue sank. Specimens were frozen in Shandon M-1 Embedding Matrix compound (Thermo Scientific, Waltham, MA) and sectioned on a freezing microtome at 40 µm. The transection segments of the spinal cords were sectioned horizontally into five sets. One set was Nissl–myelin stained and the resulting sections were examined under a microscope to confirm completeness of the transection. A second set was stained with a polyclonal antibody to 5-HT. Frozen sections were incubated at 4°C with the primary antibody (diluted 1:40,000 Immunostar, Stillwater, MN) for 16 hr, with biotinylated goat anti-rabbit IgG for 2 hr, and with avidin-biotinylated horseradish peroxidase complex for 2 hr, as specified by the manufacturer (ABC Standard Kit; Vector Laboratories, Burlingame, CA). Peroxidase reactivity was visualized with 0.05% diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05 mM Tris buffer.
Abc standard kit
Manufactured by Vector Laboratories
Sourced in United States
The ABC Standard Kit is a laboratory equipment product offered by Vector Laboratories. It is designed for general laboratory applications. The kit includes essential components necessary for various experimental procedures. The specific details and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Shandon m 1 embedding matrix compound, by Thermo Fisher Scientific (1 mentions)
Hq silver, by Nanoprobes (1 mentions)
Goat anti runx2, by Santa Cruz Biotechnology (1 mentions)
Biotinylated goat anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Cryostat, by Leica (1 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Bx50f4, by Olympus (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
5 protocols using abc standard kit
1
Histological Analysis of Spinal Cord Transection
2
Immuno-Electron Microscopy of Microglia and Amyloid
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For immuno-electron microscopy OHSCs were washed in 50 mM TBS and blocked in 20% normal goat serum (NGS) in TBS for 1 h. Subsequently, the cultures were incubated with primary antibodies rabbit anti-Iba1 (1:200) and mouse anti-6E10 (1:100) diluted in TBS containing 2% NGS overnight at 4 °C. After 1 h of washing in TBS, the OHSCs were incubated with a biotinylated anti-rabbit (for Iba1, 1:100) and a 1.4 nm gold-coupled anti-mouse secondary antibody (for 6E10; 1:100) in TBS containing 2% NGS overnight at 4 °C. OHSCs were rinsed in TBS and then fixed for 10 minutes in a 1% GA solution. Tissue-bound gold particles were enlarged using a silver intensification kit (HQ-Silver, Nanoprobes, USA). Next, sections were washed in double-destilled water for 10 min and 50 mM TB for 1 hr. Visualization of antibody binding by diaminobenzidine (DAB) staining was performed using the ABC Standard Kit (Vector Laboratories, Burlingame, USA) with DAB and H2O2 as substrates in accordance with the manufacturer’s suggestions. The sections then underwent osmification for 40 min in a solution of 0.5% OsO4 and 6.86% sucrose.
3
Osteoblastic Differentiation Assessment via Runx2 IHC
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To assess osteoblastic differentiation, we conducted an immunohistochemical test by utilizing a primary polyclonal goat antibody against Runx 2 (Goat anti-Runx2—Santa Cruz Biotechnology, SC8566, Santa Cruz, CA, USA) [25 (link)]. The team employed the immunoperoxidase detection method and amplified the signal from the reaction through incubation in avidin and biotin (ABC standard kit, Vector Laboratories, Burlingame, CA, USA). For the reaction revelation, we used Diaminobenzidine (Dako Laboratories, Santa Clara, CA, USA) and performed counterstaining with Harris hematoxylin. After dehydration through xylene, the researchers mounted coverslips over the sections to evaluate and utilize a scoring system in accordance with dos Santos Pereira et al. [26 (link)], which was performed by a single evaluator who had been previously calibrated. Scores were in the range of 0 (absence of staining), 1 (low staining), 2 (moderate staining), and 3 (intense 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.
5
Immunohistochemical Staining of Iba1+ Microglia
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Under deep anesthesia with the M/M/B anesthetic mixture, mice were perfused through the ascending aorta with 0.9% NaCl followed by 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.4). The brains were carefully dissected out of the skull and immersed in 10% to 30% sucrose solution for cryoprotection. Coronal sections of 50 μm thickness were prepared using a cryostat (Leica Biosystems, Wetzlar, Germany). The sections were treated with 0.3% Triton X followed by 3% H 2 O 2 for 10 min. After rinsing in phosphate buffered saline (PBS), the sections were incubated with 10% bovine serum albumin (Thermo Fisher Science, Waltham, MA, USA) for 1 h, followed by incubation with rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) antibody (1:1000, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 4 °C overnight. After rinsing with PBS, sections were treated with biotinylated goat anti-rabbit IgG (1:1000, Vector Laboratories, Burlingame, CA, USA) for 2 h, rinsed in PBS, and transferred to an ABC Standard kit (Vector Laboratories) for 2 h. The slides were rinsed in PBS again and stained with 3,3'-diaminobenzidine tetrahydrochloride (Dojin Glocal Co. Ltd., Kumamoto, Japan). The slides were observed under a light microscope (BX50F4; Olympus Optical Co., Tokyo, Japan), and the images were captured using a chargedcoupled device (CCD) camera (DP71, Olympus Optical Co.).
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