Fixed tissue was sectioned on a vibratome (Leica Biosystems, Buffalo Grove, IL) at 50 μm. Sequential sections were collected and stored in PBS with 0.02% NaN3 until used. CAA was visualized by immunohistochemistry for Aβ1–40 (Invitrogen, Camarillo, CA, 1:5000) as described previously[31 (link)]. Briefly, free-floating sections were pretreated with 90% formic acid for 4 min and then incubated overnight with the primary antibody, incubated with anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA). The signal was amplified and visualized with an avidin-biotin complex peroxidase kit (Vector Laboratories, Burlingame, CA), and 3,3’ diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA). Sections were mounted on glass slides and coverslipped with Depex mounting media.
Anti rabbit secondary antibody
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Anti-rabbit secondary antibody is a laboratory reagent designed to detect and bind to primary antibodies raised against rabbit antigens. This secondary antibody is conjugated with a detection label, such as a fluorescent dye or an enzyme, which allows for the visualization and quantification of the target antigen in various immunoassays and immunohistochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin peroxidase complex, by Vector Laboratories (3 mentions)
Vibratome, by Leica Biosystems (2 mentions)
Anti mouse secondary antibody, by Vector Laboratories (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Anti acetyl histone h3, by Cell Signaling Technology (2 mentions)
Anti aldh1, by BD (2 mentions)
Abc elite kit, by Vector Laboratories (2 mentions)
Rabbit anti c fos antibody, by Santa Cruz Biotechnology (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Aβ1 40, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Dab peroxidase substrate, by Merck Group (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Actin antibody, by Abcam (1 mentions)
Dkk3 antibody, by Santa Cruz Biotechnology (1 mentions)
Smad4 antibody, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Anti mbp antibody, by Santa Cruz Biotechnology (1 mentions)
27 protocols using anti rabbit secondary antibody
1
Immunohistochemical Visualization of CAA
2
Immunohistochemistry of FoxO1 in Uterine Tissue
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Uterine tissues fixed in 5% (v/v) formaldehyde (Merck, USA) for 24 h were dehydrated through a graded ethanol series and embedded in paraffin. Five micrometer cut uterine sections were taken. After deparaffinization and rehydration, citrate buffer (pH 6.0) was used for antigen retrieval using microwave. Subsequently, the slides were washed in PBS and endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 15 mins at room temperature. After washing with PBS, the sections were treated with ultra V block (Thermo, UK; cat no:TA-125-UB,) and incubated with rabbit FoxO1 primary antibody (1:100 dilution; Cell Signaling, USA; cat no:2880S) overnight at 4°C. Next day, after washing out the primary antibody, slides were incubated for 60 min at room temperature with anti-rabbit secondary antibody (Vector, USA; BA-1000, 1/500 μL,) followed by incubation with horseradish peroxidase conjugated Streptavidin (Thermo Scientific, UK; TS-125-HR) for 30 min at room temperature. All incubation steps were performed in a humidified chamber to avoid dehydration of the slides. Positive immunoreactions were visualized with diaminobenzidine (DAB)-peroxidase substrate (Sigma; cat no:D4168) and counterstaining was performed with hematoxylin. FoxO1 expression was evaluated and photographed under a Zeiss (Oberkochen, Germany) light microscope.
3
Western Blot Analysis of DKK3 and SMAD4
4
Immunohistochemical Analysis of Striatum and Corpus Callosum
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An average of six sections within the striatum spanning from Bregma 1.20 mm to 0.96 mm and six sections within the corpus callosum spanning from Bregma 0.84 mm to 0.60 mm were obtained from each brain. Tissue sections were incubated overnight with rabbit anti-GFAP antibody or anti-MBP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500.
The sections were then incubated with anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). The sections were subsequently incubated with avidin- biotin-peroxidase complex (1:100, Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution containing 0.02% 3, 3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2 in 50 mM Tris-HCl (pH 7.6) for approximately 5 min. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount® (Fisher Scientific, New Jersey, NJ, USA).
The sections were then incubated with anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). The sections were subsequently incubated with avidin- biotin-peroxidase complex (1:100, Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution containing 0.02% 3, 3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2 in 50 mM Tris-HCl (pH 7.6) for approximately 5 min. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount® (Fisher Scientific, New Jersey, NJ, USA).
5
Spinal Cord Injury Immunohistochemistry
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On days 1, 3, and 7 postinjury, 3 mice from each group were sacrificed by transcardial perfusion with 0.01 M PBS, followed by 4% paraformaldehyde in PBS. The entire spinal cord was isolated from the vertebral column, fixed for 2 h, soaked overnight in PBS, and cryoprotected in 30% sucrose. Tissue blocks centered on the SCI epicenter (3-mm blocks for transectional sections) were frozen on dry ice and sectioned at the transectional plane (10-µm thickness for transectional slices). Bovine serum albumin (BSA) was used to block nonspecific binding, and the sections were incubated with anti-SIRT2 monoclonal antibody (Cell Signaling Technology, MA, USA) according to the manufacturer's instructions at 4°C overnight and then with anti-rabbit secondary antibody (Vector, CA, USA) followed by incubation in ABC (ABC Kit, Vector) at 37°C for 30 min. The sections were then visualized by reacting with diaminobenzidine (DAB; Vector).
6
Immunohistochemical Staining Protocols
7
Immunofluorescence and Immunohistochemistry Analysis
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Antibodies used in the present study were: guinea pig anti-K14 (RDI-Fitzgerald, Acton, MA), mouse anti-Rac1, rat anti-CD45, anti-BrdU FITC (BD Bioscience, San Jose, CA), mouse anti-V5 (Invitrogen, Grand Island, NY), mouse anti-GAPDH (Cell Signaling Technology, Danvers, MA), rabbit anti-α-smooth muscle actin (αSMA) (ABcam, Cambridge, UK), and rabbit anti-actin (Santa Cruz Biotechnology). For immunofluorescence (IF), Alexa Fluor 594 (red) or 488 (green) conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were used. For αSMA immunohistochemistry (IHC), anti-rabbit secondary antibody (Vector Labs, Burlingame, CA) was used. All slides were mounted in Fluoromount G (Southernbio-tech, Birmingham, AL) with coverslips before microscopic examination.
8
Immunohistochemical Analysis of Iba-1 in Tissue
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Tissue was sectioned on a vibratome (Leica Biosystems, Buffalo Grove, Illinois, USA) at 50 μm. Sequential sections were collected and stored in PBS with 0.02% sodium azide until used. Following standard immunohistochemistry protocols and using Iba‐1 antibody (Abcam, Cambridge, Massachusetts, USA; 1:800) the sections were incubated in the primary antibody overnight and followed by incubation in an anti‐rabbit secondary antibody (Vector Laboratories, Burlingame, California, USA). This was followed by amplification of the signal using an avidin‐biotin complex peroxidase kit, and 3,3′ diaminobenzidine substrate kit. After immunohistochemistry, each tissue section was mounted on a glass slide and coverslipped with Depex mounting media. All tissue sections were immunostained within a single experiment to reduce variability.
9
Immunohistochemical Analysis of NK Cell Markers
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Frozen tumor tissue sections (5 μm) were fixed in zinc-buffered formalin (Z-fix) for 15 min. Tissue sections were permeabilized and dehydraded in −l20°C acetone for 5 min. Endogenous peroxidase was quenched with 3% hydrogen peroxide in PBS for 10 min. After washing with water, sections were incubated with protein block (Dako), and then with either anti-NK cell-specific anti-CD49b-DX5 clone (for ZR-75-1) or -HMα2 clone (MDA-MB-231) (eBioscience) or anti ISG15 antibodies overnight at 4°C. Sections were then incubated with anti-rabbit secondary antibody (Vector laboratory) (for ZR-75-1), and anti-hamster-biotin (Invitrogen) (for MDA-MB-231) for 45 min. After washing with PBS, slides were incubated with Streptavidin-HRP (Dako) (1:500) for 30 min, followed by Diaminobenzidine (DAB) substrate for 5 min. Slides were finally washed with distilled water, dehydrated in ethanol series, cleared in xylene, and covered with Permount. Sections were photographed under Nikon E600 fluorescent microscope (Nikon Instruments Inc.) with 40X (for ZR-75-1) and 20X (for MDA-MB-231) magnification.
10
Immunoperoxidase Labeling of Tyrosine Hydroxylase
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The immunoperoxidase labeling protocol was a slight modification of that reported earlier (Vidyadhara et al., 2016 (link)). Briefly, the endogenous expression of peroxidase was quenched using 0.1% H2O2 in 70% methanol, followed by blocking of non-specific staining by 3% buffered solution of bovine serum albumin for 4 h at room temperature. The sections were then incubated with the rabbit polyclonal anti-TH antibody (1:800, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by anti-rabbit secondary antibody (1:200 dilution; Vector Laboratories, Burlingame, CA, USA). The tertiary labeling was performed using avidin–biotin complex solution (1:100, Elite ABC kits; Vector Laboratories, Burlingame, CA, USA). The staining was visualized using 0.05% solution of DAB, in 0.1 M acetate imidazole buffer (pH 7.4) with 0.1% H2O2. Phosphate buffered saline (0.01 M) containing 0.3% Triton X-100 (0.01 M PBST, pH 7.4) was used as both diluent and washing buffer. Appropriate negative controls were processed identically.
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