Biotinylated anti rabbit igg

Manufactured by Vector Laboratories

Sourced in United States, Japan, Canada

Biotinylated anti-rabbit IgG is a secondary antibody used in a variety of immunochemical techniques. It is specifically designed to bind to rabbit primary antibodies, allowing for detection and visualization of target antigens.

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102 protocols using biotinylated anti rabbit igg

Immunohistochemical Analysis of MCH Neurons

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Immunohistochemical analyses were carried out as previously described [31 (link)33 (link)]. Briefly, mice were anesthetized with a mixture (3.5:1) of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (23.3 mg/ml) at a dose of 2.5 µl/g body weight. They were then perfused with 4% paraformaldehyde by a trans-cardiac method, and brains were isolated. Brains were post-fixed further in the same solution overnight at 4℃. Brains were coronally cut into 40-µm-thick sections with a vibratome (Leica VT 1000S; Leica Instruments, Nussloch, Germany). These sections in a floating state were incubated with 4% bovine serum albumin in PBS with Tween-20 (PBST) for 1 h, then reacted with the primary antibody at 4℃ overnight. The sections were washed with PBST, reacted with the secondary antibody, and visualized with an ABC Elite kit (PK-6200, Vector Laboratories, Burlingame, CA, USA) for DAB staining.
Anti-MCH was purchased from Phoenix Pharmaceuticals (#H-070-47; 1:500; Germany), while the secondary antibody, biotinylated anti-rabbit IgG, was purchased from Vector Laboratories (#BA-1000; 1:200, Burlingame, CA, USA). DAB-stained images were analyzed with an Olympus BX 51 microscope equipped with a DP71 camera and MetaMorph Microscopy Automation & Image Analysis software (Molecular Devices, Sunnyvale, CA, USA).

Kim T.K, & Han P.L. (2016). Physical Exercise Counteracts Stress-induced Upregulation of Melanin-concentrating Hormone in the Brain and Stress-induced Persisting Anxiety-like Behaviors. Experimental Neurobiology, 25(4), 163-173.

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Immunohistochemical Analysis of Xenograft and PDX Models

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Preparation of paraffin-embedded tissue sections and immunohistochemical analyses of PC3 and V16A xenografts and NEPC PDXs were carried out at histology core facility of Princess Margaret Cancer Centre. Paraffin sections at 4 μm thickness were dried at 60 °C oven for 2 hours before staining. The immunohistochemistry (IHC) was performed according to the manufacture’s guidelines using BenchMark XT-an automated slide strainer (Ventana Medical System) with standard antigen retrieval (CC1, pH 8.0, #950-124). For PIMO staining, 60 mg/kg PIMO HCl (Hypoxyprobe™-1, Hypoxyprobe) was intraperitoneal injected into tumor bearing mice and tumors were collected 60 minutes later. Mouse monoclonal anti-PIMO antibody (MAb1, Hypoxyprobe, 1:400 dilution), rabbit polyclonal anti-CA9 antibody (NB100-417, Novus, 1:1000 dilution), rabbit polyclonal anti-SYP antibody (ab32127, Abcam, 1:1000 dilution) and rabbit polyclonal anti-CHGA antibody (ab15160, Abcam, 1:1000 dilution) were used for immunohistochemistry. Biotinylated anti-rabbit IgG (Vector, BA-1000) was added to slides at 1:200 for 12 minutes. The primary-secondary antibody complex was then visualized with Ventana iView DAB Detection Kit (#760-091). The slides were counterstained with Harris hematoxylin, dehydrated in graded alcohol, cleared in xylene and coverslipped in Permount.

Guo H., Ci X., Ahmed M., Hua J.T., Soares F., Lin D., Puca L., Vosoughi A., Xue H., Li E., Su P., Chen S., Nguyen T., Liang Y., Zhang Y., Xu X., Xu J., Sheahan A.V., Ba-Alawi W., Zhang S., Mahamud O., Vellanki R.N., Gleave M., Bristow R.G., Haibe-Kains B., Poirier J.T., Rudin C.M., Tsao M.S., Wouters B.G., Fazli L., Feng F.Y., Ellis L., van der Kwast T., Berlin A., Koritzinsky M., Boutros P.C., Zoubeidi A., Beltran H., Wang Y, & He H.H. (2019). ONECUT2 is a driver of neuroendocrine prostate cancer. Nature Communications, 10, 278.

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Immunohistochemical Detection of Cleaved Caspase-3

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Paraffined sections were rehydrated and washed in 0.01-M phosphate buffered saline (PBS). Then, they were incubated over night at 4°C with a rabbit anti-cleaved caspase-3 antibody (Cell Signalling Technology, Inc., Danvers, USA) at 1∶300 dilution. Then, ocular sections were washed in PBS and incubated with the specific secondary antibody biotinylated anti-rabbit IgG (1∶100) (Vector Laboratories, Burlingame, USA). Once washed in PBS, a streptavidin Alexa Fluor 488 conjugate (Molecular Probes-Life Technologies, Grand Island, USA) at 1∶100 dilution was used to detect cleaved caspase-3 immunolabelling; the incubation was made over night at 4°C. Nuclear counterstaining with Hoescht stain solution (Sigma-Aldrich Chemie, Buchs, Switzerland) was performed for microscopic analysis with the laser scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Negative control was carried out by omitting the primary antibody. Specific labeling of cleaved caspase-3 antibody in mouse jejunal paraffin sections was used as positive control.

Bogdanov P., Corraliza L., A. Villena J., Carvalho A.R., Garcia-Arumí J., Ramos D., Ruberte J., Simó R, & Hernández C. (2014). The db/db Mouse: A Useful Model for the Study of Diabetic Retinal Neurodegeneration. PLoS ONE, 9(5), e97302.

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Immunohistochemical Staining of Paraffin Sections

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Four percent paraformaldehyde-fixed, paraffin-embedded sections in 5 μm thickness were deparaffinized with xylene and washed with distilled water. Tissue sections were boiled for 20 min in 10 mM citrate buffer (pH 7.4). After antigen retrievals, all sections were washed in distilled water, treated with 0.3% (vol/vol) hydrogen peroxide to quench endogenous peroxide, and then incubated with normal goat serum for 30 min. Sections were incubated for 2 h at room temperature with primary antibodies. The primary antibodies were serially detected with the appropriate biotinylated anti-rabbit IgG (Vector), avidin-biotinylated-peroxidase complex (Vector), and, finally, developed with diaminobenzidine (Vector). The sections were washed, counterstained with hematoxylin, dehydrated, and mounted.

Tian X., Gala U., Zhang Y., Shang W., Nagarkar Jaiswal S., di Ronza A., Jaiswal M., Yamamoto S., Sandoval H., Duraine L., Sardiello M., Sillitoe R.V., Venkatachalam K., Fan H., Bellen H.J, & Tong C. (2015). A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis. PLoS Biology, 13(3), e1002103.

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Immunohistochemical Analysis of cFLIP Protein

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Dissected CLs that were fixed in 10% (v/v) phosphate-buffered formalin and embedded in paraffin (Merck) were used for preparing sections. Sections (10 µm) were cut from the medial part of each CL, then mounted on glass slides coated with 3-aminapropyltrimethoxylane (silane; Sigma Aldrich). The sections were incubated with rabbit anti-human cFLIP_S/L antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100 with PBS containing 1% (w/v) BSA overnight at 4ºC. Next, the sections were washed with PBS and incubated with biotinylated anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA) for 30 min, washed three times in PBS, and then incubated using an VectaStain Elite ABC kit (Vector Laboratories) for 1 h at room temperature (26ºC). Peroxidase substrate-chromogen solution (Dako) was added to the sample for color development by observation under a light microscope (BX50, Olympus, Tokyo, Japan). The sections were then washed with distilled water, counter-stained with methylene green, dehydrated, mounted with Entellan (Merck), and then examined using a light microscope.

WONGPANIT K, & MANABE N. (2019). Expression and localization of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in corpora lutea during the estrous cycle and pregnancy in Thai swamp buffalo (Bubalus bubalis). The Journal of Reproduction and Development, 66(1), 29-33.

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Quantification of Cell Proliferation Markers

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Tissue samples were fixed with 4% formalin and embedded in paraffin. The slices were cut to 5 μm thickness. IHC was performed by blocking the rehydrated tissue sections using bovine serum overnight at 4°C, and subsequently applying the Anti-Ki67 antibody (Abcam, Cat. no. ab15580) and Anti-PCNA antibody (Abcam, Cat. no. ab92729). The sections were washed and then subjected to incubation with biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Laboratories, CA, USA). The ABC method (Vector Laboratories, CA, USA) was employed along with 3,3’- diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) as a substrate to detect the staining. The sections were visualized using an AX-80 microscope (Olympus, Tokyo, Japan), and Image J software (http://imagej.nih.gov/ij/) was employed for image analysis and quantification of positive expression. Statistical analysis was conducted using One-Way ANOVA.

Wang L., Wei C., Wang Y., Huang N., Zhang T., Dai Y., Xue L., Lin S, & Wu Z.B. (2023). Identification of the enhancer RNAs related to tumorgenesis of pituitary neuroendocrine tumors. Frontiers in Endocrinology, 14, 1149997.

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Immunostaining Protocol for GFP and GnRH

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Immunostaining was performed as previously described (Murakami et al., 2010 (link)). Sections were incubated with rabbit anti‐GFP antibody (Molecular Probes), followed by incubation in biotinylated anti‐rabbit IgG and avidin‐biotin peroxidase complex (Vector Laboratories, CA, USA). The peroxidase complex was visualized with 3‐3′diaminobenzidine. Sections were then treated in a 0.01 M citric acid solution (pH 6.0) using microwaves for 1–3 min to enhance immunoreactivity, processed for GnRH immunostaining, and finally detected with Vector SG (Vector Laboratories). Sections were counterstained with methyl green. Triple immunofluorescence was performed to characterize GFP‐labeled and GnRH‐negative cells. After antigen retrieval treatment, sections were incubated with a mixture of the following primary antibodies: goat anti‐GFP (Frontiers Institute), mouse anti‐GnRH (LRH13), mouse anti‐NeuN (Millipore), rabbit anti‐neuropeptide Y (Abcam), or rabbit anti‐calbindin D‐28k (Swant) for 24−48 h at 4°C and subsequently with a species‐specific secondary antibody conjugated to fluorescent dyes. Cell nuclei were visualized with 4′, 6′‐diamindino‐2‐phenylindole (DAPI, Sigma‒Aldrich, MO, USA).

Murakami S., Ohki‐Hamazaki H, & Uchiyama Y. (2022). Olfactory placode generates a diverse population of neurons expressing GnRH, somatostatin mRNA, neuropeptide Y, or calbindin in the chick forebrain. The Journal of Comparative Neurology, 530(17), 2977-2993.

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Immunohistochemical Analysis of VNUT in Mouse Calvaria

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Animal experiments were reviewed and approved by the Kyushu Dental University Animal Care and Use Committee (#18‐003). Wild‐type mouse pups on a C57BL/6J genetic background were sampled at postnatal day 5. Calvaria bones were fixed with 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany) in PBS, dehydrated through an ethanol series, embedded in paraffin, and cut into 4‐µm frontal sections [32]. Immunostaining was performed with anti‐VNUT rabbit polyclonal antibody (1 : 50 dilution). This antibody against rat or mouse VNUT was generated in rabbits using synthetic peptides corresponding to residues 5–19, RSSLMQPIPEETRKT [24] or anti‐VNUT guinea pig polyclonal antibody (1 : 50 dilution, ABN83; Sigma‐Aldrich), biotinylated anti‐rabbit IgG (1 : 400 dilution, PK‐6101; Vector Laboratories, Burlingame, CA, USA) or anti‐Guinea pig IgG HRP (1 : 100 dilution, NB7398; Novus Biologicals, Centennial, CO, USA), and the Vectastain Elite ABC kit (1 : 50 dilution; Vector Laboratories), Sigmafast 3,3′‐diaminobenzidine (DAB) tablets (Sigma‐Aldrich) were used for visualization of reaction products. Immunostained sections were counterstained with diluted hematoxylin. As a negative control for anti‐VNUT rabbit polyclonal antibody, antibody was preadsorbed with the antigenic peptide by mixing with 10 µg·µL⁻¹ peptide for 60 min at room temperature [24].

Inoue A., Nakao‐Kuroishi K., Kometani‐Gunjigake K., Mizuhara M., Shirakawa T., Ito‐Sago M., Yasuda K., Nakatomi M., Matsubara T., Tada‐Shigeyama Y., Morikawa K., Kokabu S, & Kawamoto T. (2020). VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation. FEBS Open Bio, 10(8), 1612-1623.

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Immunohistochemistry Protocol for p-p38 Detection

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The protocol for immunohistochemistry was based on the published ABC (Vectastain Elite ABC kit, Vector, CA, USA) method [38 (link)]. The DRG sections on the MAS-coated glass were preincubated in TBS containing 10% normal goat serum (NGS) for 1 hour, then incubated in primary antibody for p-p38 (1:500, Cell Signaling Technology) in the same solution for 48 hours at 4°C. The sections were washed in TBS and then incubated in biotinylated anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA) in TBS containing 5% NGS for 2 hours at 4°C, followed by incubation in avidin-biotin–peroxidase complex (Vectastain Elite ABC kit, Vector, CA, USA) for 1 hour at room temperature. The horseradish peroxidase reaction was developed in 0.1 M Tris-buffered saline, pH 7.4, containing 0.05% 3,39-diaminobenzidine tetrahydrochloride (Sigma, Steinheim, Germany), and 0.01% hydrogen peroxidase. Sections were then washed in TBS, mounted on slides, dried, and cover-slipped.

Ikeda-Miyagawa Y., Kobayashi K., Yamanaka H., Okubo M., Wang S., Dai Y., Yagi H., Hirose M, & Noguchi K. (2015). Peripherally increased artemin is a key regulator of TRPA1/V1 expression in primary afferent neurons. Molecular Pain, 11, 8.

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Optimized Immunohistochemistry for SMARCA2

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All assays were performed on formalin-fixed paraffin-embedded slides. Immunohistochemistry staining for SMARCA4 was performed as previously described.^{2 (link)} The immunohistochemistry method for SMARCA2 was optimized as follows: heat steaming with EDTA at pH 8 for 30 min resulted in epitope retrieval. Antigen retrieval followed by overnight incubation at 4 ^oC with primary antibody targeting SMARCA2 (Sigma-Aldrich, St. Louis, MO, USA; #HPA029981, dilution 1:1000). Biotinylated anti-Rabbit IgG (Vector Laboratories, Burlingame, CA, USA; #BA-100, dilution 1:1000) and ABC (Vector Laboratories; #PK-6100) were used to detect the bound antibody. Diaminobenzidine was used as the chromogen. Absence of nuclear staining in tumor cells in the presence of internal positive control (blood vessels and stromal cells) was scored as ‘loss of expression'.

Jelinic P., Schlappe B.A., Conlon N., Tseng J., Olvera N., Dao F., Mueller J.J., Hussein Y., Soslow R.A, & Levine D.A. (2015). Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type. Modern Pathology, 29(1), 60-66.

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