Survivin expression in PBMCs was detected by immunocytochemical staining using an Ultravision Detection System (Lab Vision Corporation, Newmarket Suffolk, UK), as previously described (17 (link)). Briefly, after blocking endogenous peroxidase activity, PBMCs were incubated for 1 h at room temperature with rabbit polyclonal antibody to survivin (1:1,000; Novus Biologicals). After washing, the cells were incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc., Brea, CA, USA). Nuclei were counter-stained with hematoxylin. Three replicates were performed for each sample.
Biotinylated goat anti rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Biotinylated goat anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It binds to rabbit primary antibodies, and the biotin moiety allows for further detection or signal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin horseradish peroxidase, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Ultravision detection system, by Lab Vision (1 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Pbs x, by Merck Group (1 mentions)
Pbs x, by Vector Laboratories (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Sc 8316, by Santa Cruz Biotechnology (1 mentions)
Sc 20981, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Vectastatin elite abc hrp kit, by Vector Laboratories (1 mentions)
Bx40 light microscope, by Olympus (1 mentions)
Tris based antigen unmasking solution, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Image pro plus version 6, by Media Cybernetics (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (1 mentions)
Peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions)
Ab203346, by Abcam (1 mentions)
14 protocols using biotinylated goat anti rabbit igg
1
Survivin Expression in PBMCs
2
Immunohistochemical Detection of Zif268/Egr1
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Sections were rinsed (1 × 5 min) with 0.01 M PBS to remove antifreeze solution. They were then treated for 30 min with 0.3% H2O2 to inactivate endogenous peroxidase activity. After washing (3 × 5 min) with 0.1 M PBS containing 0.15% Triton X-100 (PBS-X, Sigma-Aldrich), sections were incubated for 60 min at room temperature in 1% bovine serum albumin (BSA, Sigma Aldrich) in PBS-X. The sections were then incubated overnight at 4°C with rabbit anti-Zif268/Egr1 polyclonal antibody (1:800, dilution in BSA, Santa Cruz Biotechnology). The sections were washed with BSA-X (3 × 5 min) and incubated at room temperature for 90 min in biotinylated goat-anti rabbit IgG (1:200, dilution in PBS-X, Santa Cruz Biotechnology). They were then washed (3 × 5 min) with PBS-X and subsequently incubated for 2 h in avidin–biotin complex (1:500, dilution in PBS-X, Vector Laboratories). Sections were rinsed (3 × 5 min) in PBS-X and then stained with 3,3′-diaminobenzidine (DAB, Sigma-Aldrich). The peroxidase complex was visualized by a 5- to 10-min exposure to a chromogen solution containing 0.02% DAB with 0.3% nickel ammonium sulfate in 0.1 M PBS. The reaction was stopped by washing in PBS. The sections were mounted on gelatin-coated slides and then dehydrated and cover slipped with DPX (Sigma-Aldrich).
3
Immunohistochemical Analysis of BDNF, TrkB, and p75NTR in Rat Brain
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The protein expressions of BDNF, TrkB and p75NTR in the cortex, hippocampus, and cerebellum were detected by immunohistochemistry. Paraffin-embedded tissues were sectioned at a thickness of 10 μm and mounted onto a 1.35-μm thin polyethylene film (PALM GmbH; Wolfratshausen, Germany) overlaid on a glass slide. Sections were incubated with primary antibodies overnight at 4 °C. After wash, the sections were incubated with biotinylated goat anti-rabbit IgG (1:5000, sc-2004; Santa Cruz Biotechnologies) for 30 min. Positive staining was revealed using diaminobenzidine [SABC immunohistochemical staining kit (Cat. No. SA1025), Wuhan Boster Biological Engineering Co., Ltd., China)] according to the manufacturer’s instructions. The primary antibodies (1:1000 dilution) used were as follows: rabbit anti-rat BDNF (H-117) (sc-20,981, Santa Cruz Biotechnologies), rabbit anti-rat TrkB (H-181) (sc-8316, Santa Cruz Biotechnologies), and rabbit anti-rat p75NTR (H-92) (sc-5634, Santa Cruz Biotechnologies). The immunostained slides were imaged using Imaging-Pro Plus 6.0 [23 (link)]. Five fields were randomly selected in each section for integrated optical density (IOD) calculations. The IOD of each slice was the mean IOD of the five fields.
4
Immunohistochemical Staining of Mast Cells
5
Immunohistochemical Analysis of CD55 and CD97 Expression
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Paraffin sections (3-μm) were incubated overnight at 4°C with primary antibodies against CD55 (1:50; goat polyclonal antibody; cat. no. sc-7067; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and CD97 (1:100; rabbit polyclonal antibody; cat. no. sc-98577; Santa Cruz Biotechnology, Inc.), washed and incubated with biotinylated goat anti-rabbit IgG (1:50; goat anti-rabbit monoclonal antibody; cat. no. BA1003; Boster Biotechnology, Inc., Wuhan, China) for 30 min. Next, sections were washed three times with phosphate-buffered saline, stained with diaminobenzidine and examined by light microscopy (Olympus BX52; Olympus Corporation, Tokyo, Japan). Six randomly selected fields from each sample were examined by two pathologists, who were blinded to patient diagnosis and outcome. Areas positively expressing CD55 and CD97, and the average optical density were recorded and analyzed using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) software.
6
Histological Analysis of Joint Inflammation in Murine Arthritis Models
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Joint tissues of AIA and CIA mice were fixed in saline for 1 day and then decalcified in 5% formic acid for 2 weeks. Decalcified joints were embedded in a paraffin block. Paraffin-embedded tissue blocks were sectioned at 5 μm and then stained with hematoxylin and eosin according to the manufacturer's protocol (Sigma). For immunohistochemical analysis of macrophages, 5 μm tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. After blocking with 10% goat serum for 1 h at room temperature, sections were incubated with an anti-F4/80 macrophage marker antibody (1/50, Santa Cruz Biotechnology) overnight at 4 °C. The sections were then incubated with biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology). After washing, the sections were incubated with peroxidase-conjugated streptavidin (Vector Laboratories) for 30 min at room temperature, followed by incubation with 3′3-diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories). The sections were counterstained with hematoxylin.
7
Immunohistochemical Analysis of MT2 Receptor
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Paraffin sections were incubated in rabbit anti-MT2 primary antibody overnight at 4°C (1:500; ab203346; Abcam). The sections were then rinsed in 0.01-M PBS (pH 7.4) and incubated in biotinylated goat anti-rabbit IgG (1:300, sc-2020; Santa Cruz, CA, United States) for 2 h at room temperature. After washing, the tissues were incubated in streptavidin-horseradish peroxidase (1:300; Vector Laboratories, Burlingame, CA, United States) for 2 h at room temperature. Immunoreactivity was visualized by incubating the tissue sections in 0.01-M PBS containing 0.05% DAB and 0.003% H2O2 for 10 min in the dark. The sections were then stained with hematoxylin and mounted. Control slides without the primary antibody were examined in all cases. Immunoreactive cells presented with yellow-brown staining in the cytoplasm. The localization and distribution of immunoreactive positive materials in the hippocampus were observed using a microscope (BX51; Olympus, Japan) in accordance with a stereotaxic atlas of the mouse brain (Paxinos and Franklin, 2007 ).
8
Immunohistochemical Analysis of DHX9 Expression
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Sectioned TMA blocks (about 5 mm thick) were deparaffinized and rehydrated before immunohistochemical (IHC) staining. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide (Sigma-Aldrich) for 10 min at RT. For antigen retrieval, the slides were immersed in 10 mM citrate buffer (pH 6.0) and microwaved for 15 min. Non-specific binding was blocked with 5% normal goat serum (Invitrogen) in 1 × TBS for 10 min, followed by incubation with anti-DHX9 (1:1 000, Abcam ab26271) at 4°C overnight in a humidified chamber. The slides were sequentially incubated with biotinylated goat anti-rabbit IgG (1:100, Santa Cruz sc-2040) for 30 min at RT, followed by streptavidin-HRP conjugate for 30 min at RT. Isotope-matched human IgG was included as negative controls. For HRP enzymatic detection, 3,30-diaminobenzidine (DAB) substrate kit (Dako) was used, followed by counterstaining by Mayer’s hematoxylin. On the basis of staining intensities, the DHX9 immunoreactivity was scored as negative (0; total absence of staining), weak expression (1; faint staining in <50%, or moderate staining in <25% of tumor cells), moderate expression (2; moderate staining in > 25% to <75%, or strong staining in <25% of tumor cells) and strong expression (3; moderate staining in >75%, or strong staining in >25% of tumor cells).
9
Immunohistochemical Analysis of Spinal Cord after OEC Transplantation
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Seven days after OEC transplantation, the animals were anesthetized with 60 mg/kg sodium pentobarbital intraperitoneally and perfused with cold saline. After removing the vertebra, the spinal cord was isolated and fixed in 10% (v/v) neutral phosphate buffered formalin solution. For IHC, formalin-fixed, paraffin-embedded spinal cord sections were dewaxed and rehydrated for antigen retrieval. The sections were incubated overnight at 4 °C with the following primary antibodies: anti-Iba-1 antibody (1:50, ab139590, Abcam, Cambridge, MA), anti-PBR antibody (1:10,000, ab109497, Abcam, Cambridge, MA), and anti-IL-1Ra antibody (1:200, ab217939, Abcam, Cambridge, MA). The sections were then incubated with biotinylated goat anti-rabbit IgG for 20 min at room temperature, followed by streptavidin-peroxidase (all from Santa Cruz Biotechnology). The immunohistochemistry images were obtained under a light microscope (Leica DM4000M, Germany).
10
Immunohistochemistry of Synovial Tissues
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Synovial tissue specimens isolated from patients with RA and osteoarthritis (OA) at the time of arthroscopic biopsy or total joint replacement were stained as reported previously (Kitano et al. 2006 (link)). Briefly, synovial tissue specimens were preserved in 10% formalin, embedded in paraffin, and serially sectioned onto microscope slides at a thickness of 4 μm. Synovial tissues were stained with either anti-human c-Met antibody (1:200 dilution in PBS, rabbit IgG: Santa Cruz Biotechnology) or anti-human HGF antibody (1:200 dilution in PBS, rabbit IgG: Santa Cruz Biotechnology), washed, incubated with biotinylated goat anti-rabbit IgG (1:1000 dilution in PBS: Santa Cruz Biotechnology), washed, incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and diaminobenzidine tetrahydrochloride ([DAB], Elite kit; Vector Laboratories, Inc., Burlingame, CA, USA), and counterstained with Mayer’s hematoxylin. All patients gave informed consent, and the Institutional Medical Ethics Committee approved the study protocol.
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