Donkey antirabbit igg h l alexa fluor 594

Manufactured by Thermo Fisher Scientific

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Donkey antirabbit IgG(H+L) Alexa Fluor 594 is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassay applications.

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4 protocols using donkey antirabbit igg h l alexa fluor 594

Immunofluorescent Detection of LCN2 and Receptor

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For immunofluorescent double labelling, sections were incubated with 15% normal donkey serum at room temperature for 30 min and then incubated overnight at 4°C with primary antibodies. After washing, sections were incubated with secondary antibodies for 2 h. The primary antibodies were polyclonal goat anti-LCN2 (1:200 dilution, R&D system), polyclonal rabbit anti-SLC22A17 (marker for LCN2 receptor, 1:50 dilution, Abcam), polyclonal mouse anti-GFAP (1:400 dilution, Millipore), monoclonal mouse anti-neuronal nuclei (NeuN; 1:100 dilution, Millipore) and polyclonal goat anti-Iba1 (1:500 dilution, Abcam) or rabbit anti-Iba-1 (1:500 dilution, Wako). The secondary antibodies were donkey antigoat IgG(H+L) Alexa Fluor 594 (1:500 dilution, Invitrogen), donkey antirabbit IgG(H+L) Alexa Fluor 594 (1:500 dilution, Invitrogen), donkey antimouse IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen), donkey antirabbit IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen) and donkey antigoat IgG(H+L) Alexa Fluor 488 (1:500 dilution, Invitrogen). The double labelling was analysed using a fluorescence microscope.

Shishido H., Toyota Y., Hua Y., Keep R.F, & Xi G. (2016). Role of lipocalin 2 in intraventricular haemoglobin-induced brain injury. Stroke and Vascular Neurology, 1(2), 37-43.

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Immunofluorescence Staining of Brain Sections

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Sections were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-Block^TM Histochemical Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and then stained with the following antibodies: polyclonal rabbit anti-calbindin (1:500, CB38, Swant, Switzerland) and monoclonal mouse anti-fetuin-A (diluted 1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Sections were then incubated with donkey anti-mouse IgG (H + L) Alexa Fluor 488 or donkey anti-rabbit IgG (H + L) Alexa Fluor 594 (1:300, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. Fluoroshield^TM with DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used for nuclear staining. Stained brain sections were analyzed with an immunofluorescence microscope (Thermo Fisher Scientific, Invitrogen^TM EVOS^TM M7000 Imaging System, USA). These stained brain sections were quantified with the ImageJ software v1.52a (Bethesda, MD, USA).
Cells were plated onto sterilized glass coverslips placed in 6- or 24-well culture plates. Cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, USA), blocked with CAS-Block^TM Histochemical Reagent (ThermoFisher Scientific, Waltham, MA, USA), and stained as described above.

Yoon S., Boonpraman N., Kim C.Y., Moon J.S, & Yi S.S. (2023). Reduction of fetuin-A levels contributes to impairment of Purkinje cells in cerebella of patients with Parkinson’s disease. BMB Reports, 56(5), 308-313.

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Fibrinogen Knockdown Assay Optimization

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FGA siRNA (assay# s5115), FGB siRNA (s5119) FGG siRNAs (s5179) and negative control #1 (#4390843) were obtained from Thermo Fisher Scientific. Each siRNA concentration was titrated in 96-well tissue culture plate, in triplicates, with final concentrations ranging from 0 nm to 49 nM, in 7 nm increment. Briefly, each siRNA was dissolved in 200 μl of OPTI-MEM I Reduced Serum Medium (Gibco) mixed in equal volume of Lipofectamine RNAiMAx (Invitrogen, Carlsbad, CA, United States) with a final amount of 0.64 μl/well. siRNA and Lipofectamine were left for 5 min at room temperature for complexes to form, according to manufacturer’s instructions, and then were added to each well, mixed gently and incubated at 37°C for 48 h. At 48 h, cells were fixed in ice-cold 4% PFA. Rabbit polyclonal antibodies against human fibrinogen chain Aα antibody (20645-1-AP), fibrinogen chain Bβ antibody (16747-1-AP), and fibrinogen chain γ antibody (15841-1-AP) from Proteintech (Rosemont, IL, United States), were used. Donkey anti-Rabbit IgG H&L (Alexa Fluor^®594) (A21207, Invitrogen, Carlsbad, CA, United States) were used as a secondary antibody. Immunofluorescence was processed as described above.

Golanov E.V., Sharpe M.A., Regnier-Golanov A.S., Del Zoppo G.J., Baskin D.S, & Britz G.W. (2019). Fibrinogen Chains Intrinsic to the Brain. Frontiers in Neuroscience, 13, 541.

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Microglial Activation and Regulation Mechanisms

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The primary antibodies used in this study included polyclonal goat anti‐Iba1 (Abcam, 1:400), polyclonal rabbit anti‐Iba1 (Abcam, 1:400), polyclonal rabbit anti‐heme oxygenase‐1 (Enzo, 1:500), monoclonal mouse anti‐TLR9 (Abcam, 1:400), polyclonal rabbit anti‐TLR9 (GeneTex, 1:400), polyclonal goat anti‐GFAP (Abcam, 1:400), monoclonal rabbit anti‐Darpp‐32 (Cell Signaling, 1:100), polyclonal rabbit anti YM‐1 (Abcam, 1:200), monoclonal mouse anti‐NeuN (MERCK, 1:400), monoclonal mouse anti‐Arg1 (Santa Cruz, 1:200), polyclonal rabbit anti‐CD16 (Abcam, 1:200), polyclonal rabbit anti‐iNOS (Abcam, 1:200), polyclonal rabbit anti‐CD36 (GeenTex, 1:2000), monoclonal rabbit anti‐CD204 (Abcam, 1:2000), And polyclonal rabbit anti‐Nrf2 (Proteintech, 1:2000).
The secondary antibodies used in this article for immunofluorescence staining included donkey anti‐rabbit IgG (H + L) Alexa Fluor 488, donkey anti‐rabbit IgG (H + L) Alexa Fluor 594, donkey anti‐mouse IgG (H + L) Alexa Fluor 488, donkey anti‐mouse IgG (H + L) Alexa Fluor 594, donkey anti‐goat IgG (H + L) Alexa Fluor 488, and donkey anti‐goat IgG (H + L) Alexa Fluor 594, all obtained from Invitrogen.
The drugs used in this study included CpG ODN1826 and CpG ODN2138 (InvivoGen), clodronate liposomes, control liposomes (Liposoma Technology), and brusatol (Sigma‐Aldrich).

Wei J., Dai S., Pu C., Luo P., Yang Y., Jiang X., Li X., Lin W, & Fei Z. (2022). Protective role of TLR9‐induced macrophage/microglia phagocytosis after experimental intracerebral hemorrhage in mice. CNS Neuroscience & Therapeutics, 28(11), 1800-1813.

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