Immunohistochemical analyses were done in 4 μm femur cartilage samples. Antigen retrieval was performed by incubation with 0.1% trypsin from bovine pancreas (Sigma-Aldrich, USA) in 0.1% CaCl2. Inflammatory markers were visualized using anti-metalloproteinase (MMP)-13 (R&D systems, USA, MAB511, 30 μg/ml) and anti-ciclooxigenase (COX)-2 (sc-1745; Santa Cruz Biotechnology, USA, 1/100 dilution) antibodies as previously described (30 (link)). A secondary biotinylated anti-mouse and anti-goat IgG was used respectively for detection of positive signal through a horseradish peroxidase linked to an avidin/biotin complex (ABC) (Vector Laboratories, USA) using 3,3 diaminobenzidine tetra-hydrochloride as chromogen (Dako, Denmark). Sections were counterstained with Haematoxylin, dehydrated and mounted in DPX (Merck Millipore, USA). Positive immunoreactivity was evaluated in x20 magnification photographs obtained using a Leica DM3000 LED digital micro-imaging instrument (Leica Microsystems, USA). Each image was analyzed using the Image J software (National Institutes of Health, USA) and the percentage of positive area was calculated as previously described (30 (link), 31 (link)).
Avidin biotin complex abc
Manufactured by Vector Laboratories
Sourced in United States, China
The Avidin-biotin complex (ABC) is a versatile tool used in various biotechnological and immunological applications. It is composed of the protein avidin, which has a high affinity for the small molecule biotin. This complex is widely utilized in detection and labeling techniques due to its strong and stable binding properties.
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Lab products found in correlation
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Biotinylated goat anti rabbit antibody, by Vector Laboratories (3 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Vibratome, by Leica (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Diaminobenzidine dab, by Vector Laboratories (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Microtome, by Leica (2 mentions)
Horseradish peroxidase hrp conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Antigen retrieval buffer, by Abcam (2 mentions)
Dm3000 led, by Leica (1 mentions)
Trypsin from bovine pancreas, by Merck Group (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
Mmp 13, by R&D Systems (1 mentions)
Rabbit anti cxcr4 antibody, by Abcam (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
39 protocols using avidin biotin complex abc
1
Immunohistochemical Analysis of Femur Cartilage
2
Immunohistochemical Staining of Brain Sections
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Brain sections (30 μm in thickness) were rinsed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was quenched by washing the sections in a solution of PBS with 3% H2O2 and 10% methanol for 15 minutes. After subsequent rinsing in PBS, blocking was achieved by incubation in a solution containing 5% normal donkey serum (NDS) in 0.25% Triton X-100 in PBS (Tx/PBS) for 1 hour. Thereafter, sections were incubated with a mouse anti-neuronal Nuclei (NeuN) antibody (diluted at 1:1000, Merck Millipore, Billerica, MA, USA) or a rabbit anti-CXCR4 antibody (diluted at 1:200; Abcam, Cambridge, UK) at 4°C in 5% NDS in Tx/PBS overnight. The next day, sections were rinsed in 1% NDS in Tx/PBS followed by incubation with respective biotinylated secondary antibodies in 2% NDS in Tx/PBS at room temperature for 90 minutes. Sections were washed with Tx/PBS and incubated with avidin-biotin-complex (ABC; Vector Laboratories, Burlingame, CA, USA) for 1 hour. Following this incubation, the sections were rinsed in PBS. The ABC reaction was visualized using NiDAB (Dabsafe, Saveen Werner AB, Limhamn, Sweden) with the addition of NiCl2 and a mixture of 3% H2O2 in H2O. Thereafter, sections were rinsed in PBS, mounted on glass slides, dried overnight, dehydrated, treated with xylene and then cover-slipped with Pertex (Histolab AB, Västra Frölunda, Sweden).
3
Alzheimer's Disease Histology Protocol
4
Immunohistochemistry for GFP in Mouse Brain
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Frontal sections (50 μm) of mouse brain were generated using a freezing microtome and maintained in 100 mm PB until use. All washes were done in 100 mm PB for 10 min at RT. Briefly, after three washes free floating sections were transferred to block buffer [5% normal serum (Vector Laboratories) and 0.3% Triton X-100 (Sigma) in 100 mm PB] supplemented with an additional 5% normal serum for 1 h at RT and then incubated overnight at 4°C in block buffer containing primary antibody. In order to visualize GFP marker expression in Gli1-CreERT2 mice, we performed immunohistochemistry using a polyclonal rabbit antibody against GFP protein (Thermo Fisher, catalog #A-11122, RRID: AB_221569 ). After three washes, tissue was transferred to block buffer containing biotinylated secondary antibody for 1 h at RT, washed, then incubated in avidin-biotin complex (ABC; Vector Laboratories) in 100 mm PB for 1 h at RT. Finally, tissue was washed three times, and antibody labeling was visualized by reaction with the chromagen diaminobenzidine (DAB; Sigma). Sections were mounted onto Superfrost Plus microscope slides (Fisher Scientific), defatted in chloroform (Sigma), dehydrated through graded series of alcohol dilutions (50–100%), cleared in 100% xylene (Sigma) and finally coverslipped using Permount (Fisher Scientific) mounting media.
5
Myricetin Modulates Inflammation Signaling
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Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). Phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), LPS, nitric oxide and myricetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit PGE2 and phosphoTracer ERK1/2 (pT202/Y204), p38 MAPK (pT180/Y182), and JNK1/2/3 (pT183/Y185) ELISA kits were purchased from Abcam (ab119674, Cambridge, UK). TNF-α and IL-1β were obtained from R&D systems (Minneapolis, MN, USA). iNOS and COX-2 were obtained from cell signaling (Beverly, MA, USA). Rabbit anti-Ionized calcium binding adaptor molecule 1 (IBA-1) was obtained from Wako Pure (019-19741, Tokyo, Japan). Biotinylated goat anti-rabbit antibody and avidin-biotin complex (ABC) were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). All other reagents used were of guaranteed or analytical grade.
6
Quantification of Motor and Sensory Neurons in Twitcher Mice
7
Immunostaining of Neonatal and Adult Brains
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For VIP immunostaining, brains of mixed-sex PN0 pups were harvested at 2 h after light onset, immersion-fixed in 4% paraformaldehyde at 4°C for 6 h, cryoprotected in 30% sucrose, coronally sectioned at 20-μm thickness using a cryostat, and thaw-mounted onto gelatin-coated slides. For cFos immunostaining, adult brains described above were cryosectioned at 50-μm thickness, and floating sections were collected into phosphate-buffered saline (PBS). IHC for VIP was performed using an anti-VIP antibody [1:1000; a gift from Dr. Dick Swaab (21 –23 (link))], and IHC for cFos was performed using an anti-cFos antibody [1:400; Santa Cruz Biotechnology, Dallas, TX, USA (24 (link)–26 (link))]. Sections were incubated in the primary antibody for 1 week (for VIP IHC) or 48 h (for cFos IHC). The sections were then incubated sequentially with a secondary biotinylated donkey anti-rabbit antibody, avidin–biotin complex (ABC; Vector Labs, Burlingame, CA, USA), and reacted with diaminobenzidine with (cFos IHC) or without (VIP IHC) 0.05% nickel enhancement for color detection. After the color reaction, floating sections were washed and mounted onto gelatin-coated slides. All slides were counterstained with 1% methyl green, a nuclear stain, to visualize the perimeters of the SCN before dehydration and coverslipping.
8
Quantification of Midbrain Dopaminergic Neurons
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The procedure was performed as previously described14 (link), with some modifications. Briefly, the brains were removed, fixed for 48 h in 4% paraformaldehyde (PFA), rinsed and transferred to 0.4% PFA. After fixation, the brains were cut on a vibratome (Leica, Germany) into 30-μm sections. Sections from the midbrain including the SN/VTA were incubated overnight at 4 °C with primary anti-tyrosine hydroxylase (TH) (1:500, Millipore, USA, cat no AB1542) antibody. Visualization of antigen-bound primary antibody was carried out using a proper biotinylated secondary antibody followed by incubation with the Avidin/Biotin Complex (ABC; Vector Laboratories, USA) and diaminobenzidine staining (DAB; Sigma, USA). Stained sections were acquired and analyzed under a light microscope (Nikon Eclipse50i, Japan) equipped with a camera and NIS Elements software. Quantification of TH-positive cells (TH+) was performed manually by counting all TH+ cells on adjacent sections from 6 animals of each genotype/treatment in a single-blind experiment.
9
Immunohistochemical Staining of Brain Sections
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Brain sections (thickness 30 µm) from 4% paraformaldehyde-perfused animals were rinsed in phosphate buffered saline (PBS). Endogenous peroxidase activity was quenched by washing the sections in a solution of PBS with 3% H2O2 and 10% methanol for 15 min. After subsequent rinsing in PBS, blocking was achieved by incubation in a solution containing 5% normal donkey serum (NDS) in 0.25% Triton X-100 in PBS (Tx/PBS) for 1 h. Thereafter, sections were incubated with a mouse anti neuronal nuclei (NeuN) antibody (diluted at 1:1000, Merck Millipore, Billerica, MA, USA) at 4 °C in 5% NDS in Tx/PBS overnight. The next day, sections were rinsed in 1% NDS in Tx/PBS followed by incubation with respective biotinylated secondary antibodies in 2% NDS in Tx/PBS at room temperature for 90 min. Sections were washed with Tx/PBS and incubated with avidin–biotin-complex (ABC) (Vector Laboratories, CA, USA) for 1 h. Following this incubation, the sections were rinsed in PBS. The ABC reaction was visualized using NiDAB (DAB (Dabsafe, Saveen Werner AB, Sweden)) with the addition of NiCl2 and a mixture of 3% H2O2 in H2O. Thereafter, sections were rinsed in PBS, mounted on glass slides, dried overnight, dehydrated, treated with xylene and then cover-slipped with Pertex. (Histolab AB, Sweden).
10
Immunohistochemistry for Phosphohistone 3B
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Sections were rehydrated using standard protocols and heated in 10 mM Sodium Citrate solution (pH 6). Endogenous peroxidases were blocked by incubating in 3% H2O2 and 10% MeOH in PBS for 15 min. Sections were permeabilised in 0.2% Triton X-100 (Sigma-Aldrich) in PBS (PBT2) for 5 min and blocked using 10% heat-inactivated normal goat serum and 2% gelatin in PBT2 for 1 h. Sections were incubated in 5% GS in PBT2 containing primary antibody (rabbit anti-phosphohistone 3B (Cell Signaling (#9701), 1/100) overnight at 4 °C. After incubation with primary antibody, sections were incubated in biotinylated anti-rabbit immunoglobulin secondary antibody (Dako (#E0432), 1/200) in 5% goat serum in PBT2. Samples were washed in PBS and incubated with avidin/biotin complex (ABC, Vector) in PBS for 1 h. Sections were developed using 0.025% DAB and 0.03% H2O2 in PBS for 10 min, counterstaining using Ehrlich’s Hematoxylin solution and mounted in DPX (Sigma-Aldrich). Images were acquired on a Nikon 80i microscope equipped with a Nikon 5 M pixel Nikon DS digital camera. Images were processed using Adobe Photoshop and Illustrator.
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