biotinylated secondary antibody  (Abcam)

Journal: Cell Death & Disease

Article Title: Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson's disease

doi: 10.1038/cddis.2014.333

Figure Lengend Snippet: RTP801 is elevated in parkin knockout mouse brains. Whole-brain lysates from five 8-month-old PaKO mice and five wild-type littermates (WT) were analyzed by western immunoblotting. Membranes were incubated with anti-RTP801 and anti-parkin antibodies, and subsequently reprobed with anti- α -actin antibody as a loading control. Representative immunoblots are shown along with the densitometries representing values as mean ± S.E.M. of at least three independent membranes including all the samples. Student's t -test with Welch's correction, * P

Article Snippet: Monoclonal mouse antibody anti-parkin used for WB and immunohistochemistry was purchased from Abcam (Cambridge, UK).

Techniques: Knock-Out, Mouse Assay, Western Blot, Incubation

Journal: Cell Death & Disease

Article Title: Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson's disease

doi: 10.1038/cddis.2014.333

Figure Lengend Snippet: RTP801 is poly-ubiquitinated by parkin E3 ligase and degraded by the proteasome. ( a ) RTP801 is degraded by the proteasome. HEK293 cells were exposed for 4 h to 1 μ M epoxomycin or 50 μ M chloroquine, and cell lysates were analyzed by western immunoblotting for RTP801 and α -actin (loading control). ut = untreated. ( b ) RTP801 is poly-ubiquitinated by parkin prior to proteasomal degradation. HEK293 cells were co-transfected with pCMS-eGFP RTP801, HA-ubiquitin and one of the pRK5-myc constructs, either empty or containing parkin WT or parkin ΔR2. Twenty-four hours later, cultures were exposed to epoxomycin for 3 h prior to harvesting. RTP801 was immunoprecipitated (IP) and immunocomplexes along with whole-cell lysates were analyzed by western immunoblotting (IB). Membranes were probed for HA, myc and α -actin as a loading control. Membranes were reprobed with anti-RTP801 antibody to confirm that RTP801 was immunoprecipitated. Non-specific mouse immunoglobulins were used as negative controls (m IgG). (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) ( c ) Parkin ubiquitinates RTP801 in a cell-free in vitro system. Recombinant parkin E3 ligase (active full-length), truncated parkin (inactive), GST-RTP801, and UbcH7 E2 enzyme, were mixed and incubated as indicated along with biotinylated ubiquitin, E1 enzyme, and ATP. RTP801 was immunoprecipitated, and immunocomplexes resolved in a western blot. The membrane was incubated with Avidin/Biotin and then incubated with chemiluminiscent peroxidase substrate solution (upper panel). The same membrane was reprobed with an anti-RTP801 antibody (lower panel). (HMW Ub-RTP801, high molecular weight ubiquitinated RTP801) ( d ) Ectopic parkin physically interacts with ectopic RTP801. HEK293 cells were co-transfected either with the pCMS-eGFP and pEGFP-C2 empty vectors or pCMS-eGFP RTP801 along with pEGFP-C2-parkin. Twenty-four hours later, cultures were treated with the cross-linking agent DSP for 2 h at 4°C, prior to harvesting. After RTP801 immunoprecipitation, the samples were analyzed by western immunoblotting for parkin to detect the interaction, and for RTP801 as an IP control. ( e and f ) Reciprocal co-immunoprecipitation indicates interaction of endogenous parkin and RTP801 in cells. Neuronal PC12 cells were treated with or without MG132 for 5 h as indicated, lysed and subjected to immunoprecipitation (IP) with control antibody (GFP), and either ( e ) anti-parkin or ( f ) anti-RTP801 antibodies. Immunoprecipitates were analyzed by western immunoblotting with anti-parkin and anti-RTP801 as indicated. In ( e ) all samples were analyzed on the same blot, but irrelevant intervening lanes were removed. In the right panel ( f ), the membrane was probed with a light chain-specific secondary antibody to diminish overlap of signal with co-immunoprecipitated RTP801. In each panel ( a , b , c , d , e and f ), a representative western blot is shown from a pool of at least two independent experiments. Specific bands are pointed out by arrows

Article Snippet: Monoclonal mouse antibody anti-parkin used for WB and immunohistochemistry was purchased from Abcam (Cambridge, UK).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Molecular Weight, In Vitro, Recombinant, Incubation, Avidin-Biotin Assay

Journal: Veterinary World

Article Title: Detection of Toxoplasma gondii in cat’s internal organs by immunohistochemistry methods labeled with-[strept] avidin-biotin

doi: 10.14202/vetworld.2017.1035-1039

Figure Lengend Snippet: Toxoplasma positive immunohistochemistry staining of cat with labeled-(Strept) avidin-biotin method: (a) Necrosis is present in kidney and (b) ileum (arrow) (brown color is indicating the positive result of immunohistochemistry) (scale bar=30 µm).

Article Snippet: Slides were incubated with primary polyclonal anti-T. gondii antibody (rabbit) primary antibody (Abcam, USA) diluted 1/100 at 37°C for 32 min and biotinylated secondary antibody (Abcam, USA).

Techniques: Immunohistochemistry, Staining, Labeling, Avidin-Biotin Assay

Journal: Developmental biology

Article Title: Perichondrium phenotype and border function are regulated by Ext1 and heparan sulfate in developing long bones: A mechanism likely deranged in Hereditary Multiple Exostoses

doi: 10.1016/j.ydbio.2013.02.008

Figure Lengend Snippet: Binding interactions between BMP2 and heparin are reversed by HS antagonism. (A) Association–dissociation curves of rhBMP2 injected on a streptavidin chip coated with biotinylated heparin. Binding constants were calculated and are shown in

Article Snippet: Following rinsing, sections were then incubated with biotinylated anti-rabbit secondary antibody and the signal was visualized using a HRP/DAB detection IHC kit according to the manufacturer’s instructions (Abcam).

Techniques: Binding Assay, Injection, Chromatin Immunoprecipitation

Journal: Journal of Cancer

Article Title: Determination of the Expression of PD-L1 in the Morphologic Spectrum of Renal Cell Carcinoma

doi: 10.7150/jca.35738

Figure Lengend Snippet: Double IHC staining in HLRCC. CD4 + lymphocytes in red color, CD8 + lymphocytes in brown color. The peritumoral distribution of immune cells is seen, with not immune cells within the tumor areas. Magnification: A: 20x, B:40x.

Article Snippet: For the secondary antibody, sections were incubated with Rabbit Anti-CD4 (EPR6855; Abcam, at 1:500dil) followed by avidin-biotin complex for 15 minutes at room temperature and developed alkaline phosphatase using a kit from Vector Laboratories.

Techniques: Immunohistochemistry, Staining