Paraformaldehyde-fixed hippocampal tissues were sliced into 20-μm sections using a sliding microtome (SM1020R, Leica). The immunohistochemistry procedure was performed as previously described (Medeiros et al., 2014 (link)), including an antigen retrieval step (Tris/EDTA pH 9.0 at 97°C for 10 min). Staining with anti-METTL3 antibody (ab195352, Abcam, 1:50 dilution) was performed overnight at 4°C, followed by detection with biotinylated horse anti-rabbit IgG (Vector Laboratories), avidin-biotin complex (Vector Laboratories), and diaminobenzidine (DAB) staining. Stained hippocampal sections were imaged with a Zeiss Axio Imager Z2 microscope equipped with a 20× objective and a Metafer slide scanning platform. Quantification of the optical density (OD) of images was performed with Fiji software (ImageJ, NIH). Briefly, color deconvolution of the images was achieved using the set OD vectors for DAB and hematoxylin (H DAB). The OD value of the image presenting DAB was calculated using the formula OD = log(max intensity/mean intensity). Selected regions from CA1, CA2, CA3, and the dentate gyrus (DG) were measured separately for each section.
Biotinylated horse anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated horse anti-rabbit IgG is a secondary antibody that specifically binds to rabbit immunoglobulin G (IgG) and is conjugated with biotin. It is intended for use in various immunoassay techniques that require the detection of rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated horse anti mouse igg, by Vector Laboratories (4 mentions)
Srebp1, by BD (2 mentions)
Imagescope v 11, by Leica Biosystems (2 mentions)
F4 80, by Bio-Rad (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Ki 67 h 300, by Santa Cruz Biotechnology (2 mentions)
Novocastra streptavidin hrp, by Leica camera (2 mentions)
Aec high sensitivity substrate chromogen, by Agilent Technologies (2 mentions)
Cdk4 c 22, by Santa Cruz Biotechnology (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Ab195352, by Abcam (1 mentions)
Sm1020r, by Leica camera (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Safranin o, by Merck Group (1 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Abc peroxidase kit, by Vector Laboratories (1 mentions)
14 protocols using biotinylated horse anti rabbit igg
1
Quantifying METTL3 in Hippocampal Regions
2
Quantification of Dopaminergic Neurons
3
Chondrogenic Differentiation Assay
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A total of 2.5 × 105 passage-3 SDSCs were seeded into 15-mL centrifuge tubes and cultured in 0.5 mL of chondrogenic differentiation medium for 21 days. The pellets (n = 2) were fixed in 4% paraformaldehyde at 4 °C overnight, followed by dehydration in a gradient ethanol series, clearing with xylene, and embedding in paraffin blocks. Sections with a thickness of approximately 5 mm were stained with Alcian Blue (Sigma; counterstained with Fast Red) and Safranin O (Sigma; counterstained with Hematoxylin) for sulfated glycosaminoglycans (GAGs). For immunohistochemistry analysis, sections were probed with primary antibodies against collagen II (II-II6B3; DSHB, Iowa City, IA, USA), followed by the secondary antibody biotinylated horse anti-rabbit IgG (Vector, Burlingame, CA, USA). Immunoactivity was detected using Vectastain ABC reagent (Vector) with 3,3′-diaminobenzidine (DAB) as a substrate. Hematoxylin (Vector) served as a counterstain.
4
Immunohistochemical Staining of AT1/AT2, ACE2, Macrophages, and α-SMA
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Immunohistochemical staining was performed, as we have previously reported.10 (link) Briefly, paraffin sections were stained using rabbit anti-AT1/AT2 receptor polyclonal antibodies, and a rabbit polyclonal anti-ACE2 antibody (Santa Cruz Biotechnology), a rabbit monoclonal antibody against macrophages (CD68; EMD Millipore, Billerica, MA, USA), and a monoclonal antibody used against alpha smooth muscle actin (α-SMA; Sigma-Aldrich Co). The slides were incubated with a biotinylated horse anti-rabbit IgG or an anti-mouse IgG (Vector Laboratories, Inc, Burlingame, CA, USA), stained using the ABC peroxidase kit or ABC-AR kit (alkaline phosphatase; Vector Laboratories, Inc), and substrated with a 3,3′-diaminobenzidine tetrahydrochloride or alkaline phosphatase substrate kit (Sigma-Aldrich Co). Quality of immunohistochemistry assay was controlled by either elimination of the primary antibody or incubation of the tissue with a non-immune IgG. The intensity in expression of AT1/AT2 receptors and ACE2, as well as accumulation of macrophages and α-SMA-expressing myofibroblasts among groups, were compared using computer-assisted morphometry (ImageJ software). The final results were averaged from the eight randomized high-powered fields.
5
Immunohistochemical Analysis of nSMase2 in Rat Brain
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Six adult Wistar rats were used for this portion of the study. They were anesthetized deeply and perfused through the left cardiac ventricle with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and sectioned coronally at 100 μm using a vibratome. Sections were washed for 3 h with phosphate-buffered saline (PBS) and incubated overnight at 4 °C with an affinity-purified rabbit polyclonal antibody to nSMase2 (Santa Cruz Biotechnology, Santa Cruz, USA), diluted 1:200 in PBS. They were then incubated for 1 h in a 1:200 dilution of biotinylated horse anti-rabbit IgG (Vector, Burlingame, CA, USA), reacted for 1 h with avidin-biotinylated horseradish peroxidase complex, and treated for 5 min in 0.05% 3, 3-diaminobenzidine tetrahydrochloride solution in Tris buffer containing 0.05% hydrogen peroxide. Some of the sections were mounted on glass slides, counterstained with methyl green, and coverslipped, while the remaining sections were processed for electron microscopy.
6
Quantification of Pulmonary Proteinaceous Accumulation
7
Immunolabeling of RIP3 and MLKL
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For immunolabeling with the avidin-biotin complex method, sections were treated in 0.3% H2O2 in 0.01 M PBS (pH 7.3) for 15 minutes to inactivate endogenous peroxidase. Non-specific antibody binding was blocked by pre-incubating the sections in 5% normal horse serum (Sigma) in PBS containing 0.3% Triton X-100 (Fluka, St.Louis, MO, USA) for 1 hour at room temperature. Sections were then incubated with rabbit monoclonal anti-RIP3 (1:1000), or rabbit polyclonal anti-MLKL (1:1000) antibody at 4°C overnight, then reacted with biotinylated horse anti-rabbit IgG (1:400; Vector Laboratories Inc, Burlingame, CA, USA) for 2 hours at room temperature. After 1-hour incubation with the avidin-biotin complex reagents (1:400; Vector Laboratories Inc.), the immunoreaction product was visualized in PBS containing 0.05% 3,3′-diaminobenzidine (Sigma) and 0.03% H2O2. Finally, the RIP3 sections were counterstained by hematoxylin, dehydrated, cleared and coverslipped. These sections were observed and images were captured using a microscope (Nikon, Tokyo, Japan).
8
SREBP-1 and GLS Expression in Lung Cancer and Glioma
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Individual lung tumor and adjacent normal tissues, lung tumor tissue microarray (TMA) containing 50 paired (tumors and matched adjacent normal lung tissues) and 49 unpaired lung tumor tissues, and individual GBM tumor tissues were from the Department of Pathology at The Ohio State University. All human tissues were collected from Ohio State University Hospitals under Institutional Review Board- (IRB) and HIPPA-approved protocols, and histologically confirmed. Glioma TMA with 91 tumors was from the University of Kentucky and IRB approval was obtained at UK prior to study initiation. All samples had tested negative for HIV and hepatitis B. TMA slides were stained using SREBP-1 (BD Pharmingen, 557036, 1:20) or GLS (Abcam, #ab93434, 1:50) antibodies and then using biotinylated horse anti-mouse IgG (Vector labs, Cat#BA-2000, 1:500) or biotinylated horse anti-rabbit IgG (Vector labs, Cat#BA-1100, 1:500) antibodies. The slides were scanned using ScanScope and analyzed using ImageScope v11 software (Aperio Technologies, Vista, CA, USA). The staining intensity of tissues was graded as 0, 1+, 2+, or 3+. H-score was calculated using the following formula: H score = [1 x (%cells with 1+) + 2 x (%cells with 2+) + 3 x (%cells with 3+)] x 100.
9
Immunohistochemical Analysis of Amyloid-Beta
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The 10-μm frontal cortex slices were obtained using a freezing microtome (Leica, Nussloch, Baden-Württemberg, Germany). Brain slices were pretreated for peroxidase activity using 3% hydrogen peroxide (H2O2) in 0.1 M PBS containing 5% Triton X-100 for 20 min, and blocked with 5% normal horse serum (NHS; Vector Laboratories, Burlingame, CA, USA) for 1 h. Thereafter, selected tissue slices were incubated with primary antibody of beta-amyloid Clone 6F/3D (1:800, Code-Nr. M 0872, Dako) at 4°C overnight. Slices were again washed three times with 0.1 M PBS, incubated with biotinylated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) secondary antibody for 1 h, and then treated with biotinylated protein A and avidinbiotinylated horseradish peroxidase complexes (ABC kit, Vector Laboratories, Burlingame, CA, USA) for another 1 h at room temperature. Immunoreactive cells were visualized using 3,3′-diaminobenzidine (DAB) and urea H2O2 tablets (Sigma-Aldrich, St. Louis, MO, USA) dissolved in double distilled water. Finally, sections were mounted onto glass slides, dehydrated with a graded series of ethanol, cleared in xylene, and cover slipped.
10
Quantification of Dopaminergic Neurons
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Mouse tissues were fixed by perfusion as described and cryostat-cut sections (40 μm) were taken through the entire midbrain (Mallajosyula et al., 2008 (link)). TH+ neurons were immune-labeled by incubating the sections successively with a rabbit polyclonal anti-TH antibody (1:200) and biotinylated horse anti-rabbit IgG (1:200, Vector Laboratories) in combination with 3,3′-diaminobenzidine (DAB) reagents. Total # TH+ SNpc neurons were counted from 4 to 5 litter-mates per group using the optical fractionator method (Peng et al., 2010 (link)).
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