Goat anti rabbit secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Goat anti-rabbit secondary antibody is a laboratory reagent used to detect and quantify rabbit primary antibodies in various immunoassays. It provides a sensitive and specific means of amplifying the signal from rabbit primary antibodies, enabling researchers to accurately measure the presence and levels of their target analytes.

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Lab products found in correlation

Goat anti mouse secondary antibody, by Thermo Fisher Scientific (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Hematoxylin, by Merck Group (2 mentions) 3 3 diaminobenzidine (dab), by Biocare Medical (2 mentions) Goat anti human b7 h3 antibody, by R&D Systems (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions) Bcl 2, by Cell Signaling Technology (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Gapdh, by Abcam (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Gapdh, by Proteintech (2 mentions) Tcs sp8, by Leica (2 mentions) Ab210554, by Abcam (2 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Beyotime (2 mentions) Gallios flow cytometer, by Beckman Coulter (2 mentions) Rat monoclonal anti f4 80 clone a3 1, by Abcam (2 mentions)

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101 protocols using goat anti rabbit secondary antibody

Immunohistochemical Evaluation of B7-H3 and CDC25A

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Sections from paraffin-embedded tissues were incubated with a goat anti-human B7-H3 antibody (1:200, R&D Systems) or rabbit anti-human CDC25A antibody (1:100, Abcam) overnight at 4 °C. This step was followed by staining (45 min at room temperature) with the corresponding HRP-labeled rabbit anti-goat secondary antibody or goat anti-rabbit secondary antibody (Invitrogen). Next, the sections were visualized by staining with 3,3'-diaminobenzidine (Biocare Medical, California, USA) and counterstaining with hematoxylin (Sigma).
All sections were then reviewed blindly by two experienced pathologists (Dr. Cao and Dr. Zhan). The scoring criteria for B7-H3 and CDC25A immunostaining was using based on clinical data and adopted the semiquantitative immunoreactive score (IRS) system 17 (link). Briefly, category A (intensity of immunostaining) was scored using the following criteria: 0, negative; 1, weak; 2, moderate; and 3, strong. Category B (percentage of immunoreactive cells) was scored using the following criteria: 1, (0-25%); 2, (26-50%); 3, (51-75%); and 4, (76-100%). Final scores were calculated by multiplying the scores of categories A and B in the same section; the scores ranged from 0 to 12.

Ma Y., Wang R., Lu H., Li X., Zhang G., Fu F., Cao L., Zhan S., Wang Z., Deng Z., Shi T., Zhang X, & Chen W. (2020). B7-H3 promotes the cell cycle-mediated chemoresistance of colorectal cancer cells by regulating CDC25A. Journal of Cancer, 11(8), 2158-2170.

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Immunohistochemical Analysis of B7-H3, KIF15, and Ki67

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Sections from paraffin-embedded tissues were incubated with a goat anti-human B7-H3 antibody (1:200, R&D Systems, #AF1027), a rabbit anti-human KIF15 antibody (1:2000, Proteintech, #55407-1-AP) or a rabbit anti-human Ki67 antibody (1:500, Abcam, ab15580) overnight at 4 °C. This step was followed by staining (45 min at room temperature) with the corresponding HRP-labeled rabbit anti-goat secondary antibody or goat anti-rabbit secondary antibody (Invitrogen). Next, the sections were visualized by staining with 3,3’-diaminobenzidine (Biocare Medical, CA, USA) and counterstaining with hematoxylin (Sigma). The numbers of Ki67-positive cells and total cells were analyzed using a microscope (Leica, Buffalo Grove, USA).
All sections were then reviewed blindly by two experienced pathologists (Dr. Cao and Dr. Zhan). The scoring criteria for B7-H3 and KIF15 immunostaining were based on clinical data and adopted the semiquantitative immunoreactive score (IRS) system^{17 (link)}. Briefly, category A (intensity of immunostaining) was scored using the following criteria: 0, negative; 1, weak; 2, moderate; and 3, strong. Category B (percentage of immunoreactive cells) was scored using the following criteria: 1 (0–25%); 2 (26–50%); 3 (51–75%); and 4 (76–100%). Final scores were calculated by multiplying the scores of categories A and B in the same section; the scores ranged from 0 to 12.

Ma Y., Zhan S., Lu H., Wang R., Xu Y., Zhang G., Cao L., Shi T., Zhang X, & Chen W. (2020). B7-H3 regulates KIF15-activated ERK1/2 pathway and contributes to radioresistance in colorectal cancer. Cell Death & Disease, 11(10), 824.

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Immunofluorescence Characterization of Neuropeptide F

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We carried out whole-mount brain immunofluorescence as previously described [38 ] using an anti-NPF primary antibody at 0.05 μg/μL (RayBiotech [RB-19-001], US) and a goat anti-rabbit secondary antibody coupled to Alexa Fluor 488 (1:1000, Life technologies, US). We imaged the brains with a 20X objective on an LSM780 confocal microscope (Zeiss, Germany) and quantified staining intensities of individual cells using ImageJ [37 (link)].
To verify GAL4 line expression patterns, we crossed NPF-GAL4 and r4-GAL4 to UAS-GFP and then dissected the fat body, brain, and ventral nerve cord from 5-d-old male flies. After fixing these tissues with 4% paraformaldehyde in PBS, we stained them with a rabbit anti-GFP antibody (1:1000, A11122, Invitrogen, US) and a goat anti-rabbit secondary antibody coupled to Alexa Fluor 488 (1:1000, Life technologies, US).

Kim D.H., Shin M., Jung S.H., Kim Y.J, & Jones W.D. (2017). A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila. PLoS Biology, 15(3), e2000532.

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Western Blot Analysis of Signaling Pathways

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Total protein was extracted by 1× SDS protein lysis buffer supplemented with protease inhibitors (MCE, Monmouth Junction, NJ, USA) and phosphatase inhibitors (MCE, Monmouth Junction, NJ, USA). Total protein was separated in 12% SDS-PAGE and transferred to 0.45 μm PVDF membranes (EMD Millipore, Burlington, MA, USA). After blocking with 5% bovine serum albumin, the membranes were incubated respectively with Rabbit-anti Cdc42 (CST, Danvers, MA, USA), JNK (CST, Danvers, MA, USA), phospho-JNK (CST, Danvers, MA, USA), p44/42 MAPK (ERK1/2) (CST, Danvers, MA, USA), phospho-p44/42 MAPK (ERK1/2) (CST, Danvers, MA, USA), p38 (CST, Danvers, MA, USA), phospho-p38 (CST, Danvers, MA, USA), p65 (CST, Danvers, MA, USA), phospho-p65 (CST, Danvers, MA, USA), HSP90 (CST, Danvers, MA, USA), β-Tubulin (CST, Danvers, MA, USA) and GAPDH primary antibodies (CST, Danvers, MA, USA), then conjugated with Goat anti-Rabbit secondary antibody (Invitrogen, Gaithersburg, MD, USA). Proteins were visualized by Odyssey CLx Infra-Red Imaging system (Odyssey CLx, Lincoln, NE, USA).

Ma X., Liu H., Zhu J., Zhang C., Peng Y., Mao Z., Jing Y, & Chen F. (2022). miR-185-5p Regulates Inflammation and Phagocytosis through CDC42/JNK Pathway in Macrophages. Genes, 13(3), 468.

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Immunostaining of MKX in Cell Monolayers

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Monolayer cultures of cells were fixed in 4% (vol/vol) paraformaldehyde and subjected to immunostaining with the primary antibody, rabbit anti‐human MKX monoclonal antibody (LifeSpan BioSciences, Seattle, WA, https://www.lsbio.com/), followed with a goat anti‐rabbit secondary antibody (Invitrogen). 4′,6‐diamidino‐2‐phenylindole (DAPI) staining was used to reveal the nuclei of the cells.

Chen J., Zhang E., Zhang W., Liu Z., Lu P., Zhu T., Yin Z., Backman L.J., Liu H., Chen X, & Ouyang H. (2017). Fos Promotes Early Stage Teno‐Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon. Stem Cells Translational Medicine, 6(11), 2009-2019.

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Immunohistochemical Analysis of Liver CPS1

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Livers were removed from euthanized animals and fixed in 10% neutral buffered formalin in histology cassettes for 48 hours and then stored in 70% ethanol followed by paraffin embedding using standard procedures. For each embedded tissue, 4 μm thick cross sections were collected on microscope slides, which were subsequently used for immunohistochemistry staining procedures. The sections were deparaffinized in xylene and rehydrated in serial washes of ethanol. Antigen retrieval was performed in10 mM sodium citrate buffer pH 6.0 in a steamer for 30 min followed by cooling at RT for 20 min. Following antigen retrieval, tissue sections were permeabilized with 0.1% Triton X-100 in PBS for 10 min prior to blocking with 10% normal goat serum in PBS for one hour. Later, sections were incubated with CPS1 antibody (Abcam ab45956) at 1 μg/ml in the blocking buffer overnight at 4°C. After overnight incubation, the sections were stained with Goat anti-Rabbit Secondary Antibody (Invitrogen A-11012) for 1 hour at RT. The cell nuclei were counterstained with DAPI using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). The stained tissue sections were visualised with LSM 880 with Airyscan Confocal Microscope (Carl Zeiss, Oberkochen, Germany).

Khoja S., Nitzahn M., Hermann K., Truong B., Borzone R., Willis B., Rudd M., Palmer D.J., Ng P., Brunetti-Pierri N, & Lipshutz G.S. (2018). Conditional Disruption of Hepatic Carbamoyl Phosphate Synthetase 1 in Mice Results in Hyperammonemia without Orotic Aciduria and Can be Corrected by Liver-Directed Gene Therapy. Molecular genetics and metabolism, 124(4), 243-253.

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Heme Oxygenase-1 Signaling Pathway Analysis

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Fetal bovine serum (FBS) (Catalog # 89510-188), Dulbecco’s Modified Eagle’s Medium (DMEM) (Catalog # L0102-0500), Trypsin 0.25% w/v (Catalog # 45000-664), ALA (Catalog # BT143115-2G), ascorbic acid, cell culture grade dimethylsulfoxide (DMSO), oxalic acid, Dulbecco’s Phosphate Buffered Saline (DPBS), and LiCOR Intercept tris-buffered saline blocking buffer (Catalog # 103749-018) were purchased from VWR/Avantor. Opti-MEM (Catalog # 31-985-070) was purchased from Fischer Scientific. Lipofectamine LTX and PLUS reagent (Catalog # 15338100), Lipofectamine RNAiMax (Catalog # 13778075), and a mounting reagent with 4′,6-diamidino-2-phenylindole were purchased from Invitrogen. Succinylacetone (Catalog # D1415) was purchased from Sigma Aldrich. Digitonin and hemin chloride were acquired from Calbiochem. The following primary and secondary antibodies were used: rabbit polyclonal HO-2 antibody (Abcam ab90515); mouse monoclonal GAPDH antibody (Sigma 8795); rabbit polyclonal HO-1 antibody (Enzo Life Sciences BML-HC3001-0025); mouse monoclonal calnexin antibody (Invitrogen MA3-027); mouse monoclonal ubiquitin antibody (P4D1) (Santa Cruz Biotechnology sc-8017); anti-FLAG M2 magnetic bead (Sigma Aldrich M8823); goat anti-rabbit secondary antibody (Biotium 20064); and goat anti-mouse secondary antibody (Invitrogen SA5-35521).

Hanna D.A., Moore C.M., Liu L., Yuan X., Dominic I.M., Fleischhacker A.S., Hamza I., Ragsdale S.W, & Reddi A.R. (2021). Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells. The Journal of Biological Chemistry, 298(2), 101549.

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Quantifying α-SMA Stress Fibers in Mouse PLFs

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Mouse PLFs were cultured in 4-chamber Nunc Lab-Tek slides ± IL-17 (10 ng/ml) for 96 hours (Thermo Fisher Scientific, Norcross, GA). Cells were fixed, permeabilized, and then stained for α-SMA overnight using rabbit anti-mouse α-SMA antibody at a 1:200 dilution at 4C (Abcam, San Francisco, CA). The chamber slides were incubated with a goat anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA) and DAPI stain (Vector Labs, Burlingame, CA) and examined using an Olympus BX-41 fluorescence microscope (Olympus, Center Valley, PA). Cell count was performed from four random fields per sample (x20 magnification) from triplicate experiments. Data were expressed as percent of cells with α-SMA stress fiber formation compared with total number of cells.

Neveu W.A., Staitieh B.S., Mills S.T., Guidot D.M, & Sueblinvong V. (2019). Alcohol-induced interleukin-17 expression causes murine lung fibroblast-to-myofibroblast transdifferentiation via Thy-1 downregulation. Alcoholism, clinical and experimental research, 43(7), 1427-1438.

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SARS-CoV-2 N Protein Immunofluorescence Assay

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The cells in the 12‐well plate were washed three times with PBS at 2 and 24 h after infection, and then fixed with 4% paraformaldehyde for 30 min. Cells was permeabilized with PBST (0.1% Triton × 100) for 20 min and washed three times with PBS. Cells were then blocked with 3% goat serum for 1 h and washed three times with PBST. Then, 200 μl of N protein antibody (rabbit antibody, SinoBiological) diluted at 1:500 was added to cells, followed by incubation at 4°C overnight. Next day, cells were washed three times with PBST. Goat anti‐rabbit secondary antibody (Invitrogen A‐11020) diluted at 1:5000 was added to cells, followed by incubation for 2 h at room temperature. Cells were washed three times with PBS. The slides were mounted with anti‐fade mounting medium ProLong Gold (ThermoFisher P10144) (with DAPI) and finally imaged under a Leica TCS SP8 CARS (Leica) confocal microscope.

Yang H., Liu P., Zhang Y., Du T., Zhou Y., Lu S, & Peng X. (2022). Characteristic analysis of Omicron‐included SARS‐CoV‐2 variants of concern. MedComm, 3(2), e129.

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Visualizing MMP-14 in MDA-MB-231 Cells

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MDA‐MB‐231 cells (1.5 × 10⁴) were seeded onto Matrigel‐coated glass coverslips and cultured for 24 h in DMEM supplemented with 10% FCS at 37 °C. After two washes with PBS, cells were cultured with or without κE (50 μg·mL⁻¹) for 6 or 24 h in DMEM without FCS, then fixed in 2% paraformaldehyde, washed with PBS and incubated for 1 h at room temperature in blocking solution (PBS containing 3% bovine serum albumin). Cells were then incubated overnight at 4 °C with MMP‐14 primary antibody (rabbit anti‐human MMP‐14 antibody, clone EP1264Y, GeneTex), washed and stained for 30 min at room temperature with goat anti‐rabbit secondary antibody (Invitrogen/Thermo Fisher Scientific) conjugated to Alexa Fluor 568 and Hoescht reagent. Finally, they were mounted in FluorSave Reagent (Merck, Darmstadt, Germany) and examined using an LSM710 NLO laser scanning confocal microscope (Carl Zeiss Axio Observer). Emitted fluorescence was detected through the use of the appropriate filter set, and representative images from three separate experiments were treated and merged with imagej software (NIH, Bethesda, MD, USA).

Salesse S., Odoul L., Chazée L., Garbar C., Duca L., Martiny L., Mahmoudi R, & Debelle L. (2018). Elastin molecular aging promotes MDA‐MB‐231 breast cancer cell invasiveness. FEBS Open Bio, 8(9), 1395-1404.

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