The mouse eyeballs were fixed in Davidson’s fixation solution for 48 h for the paraffin section. Following the antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a steam bath, three washes with phosphate-buffered saline (PBS) and blocking with 5% bovine serum albumin (BSA) in PBS, the sections were incubated with anti-PPARα (Novus #NB600-636; Centennial, CO), anti-TOMM20 (abcam #ab186735; Waltham, MA), or anti-nitrotyrosine (abcam #ab61392) antibodies overnight. After three washes with PBS, the slides were incubated with Alexa Fluor 488 AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch #711-545-152; West Grove, PA) or Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch #715-585-150). The slides were mounted with Vectashield mounting buffer containing DAPI (Vector Laboratories #H-1200; Newark, CA) and photographed under a Zeiss Microscope (Observer Z1; Pleasanton, CA).
Alexa fluor 594 affinipure donkey anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United States
Alexa Fluor 594 AffiniPure donkey anti-mouse IgG is a secondary antibody conjugated with Alexa Fluor 594 dye. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (4 mentions)
Ab186735, by Abcam (1 mentions)
Ab61392, by Abcam (1 mentions)
Nb600 636, by Abcam (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
Alexa fluor 488 affinipure donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Sp5 confocal fluorescence microscope, by Leica (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Sc 271243, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Abcam (1 mentions)
Mvx10, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Nanog, by Thermo Fisher Scientific (1 mentions)
Ssea4, by Thermo Fisher Scientific (1 mentions)
Tra 1 60, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
5 protocols using alexa fluor 594 affinipure donkey anti mouse igg
1
Immunofluorescent Localization of Mitochondrial and Oxidative Markers
2
Immunohistochemical Analysis of Inflammatory Markers
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Formalin-fixed, paraffin-embedded tissue slides obtained from patients and healthy controls were heated for 30 minutes at 60°C, deparaffinized, and rehydrated. Slides were placed in pH 9 antigen retrieval buffer and heated at 125°C for 30 seconds in a pressure cooker water bath. After cooling, slides were blocked using 10% donkey serum (30 minutes). Overnight coincubation (4°C) was then performed using anti–human TNF (Abcam, catalog ab6671), anti–human CD20 (Santa Cruz Biotechnology, catalog sc-393894), and anti–human CD138 (LifeSpan Biosciences, LS-B9360-50). Slides were then washed and treated with relative fluorescence-conjugated secondary antibodies (30 minutes). The secondary antibody used with anti–human TNF was Alexa Fluor 488 AffiniPure donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories, catalog 711-545-152). The secondary antibody used with anti–human CD20 and anti–human CD138 was Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, catalog 715-585-151). Slides were prepared in mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (VECTASHIELD Antifade Mounting Medium with DAPI, H-1200, Vector Laboratories, Maravai LifeSciences). Images were acquired using a ZEISS Axioskop 2 microscope. Images presented are representative of at least 3 biologic replicates.
3
Immunofluorescence Staining and Apoptosis Assay
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Immunofluorescence (IF) staining was performed as described previously 22 (link). For double staining, the samples were incubated in a mixture of two primary and then secondary antibodies. The primary antibody information was listed in Supplementary Table 1 . DAPI-Fluoromount-G was purchased from Southern Biotech. The secondary antibodies included Alexa Fluor 594 AffiniPure donkey anti-rabbit IgG (red), Alexa Fluor 488 AffiniPure donkey anti-goat IgG (green), Alexa Fluor 488 AffiniPure donkey anti-rabbit IgG (green), Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (red) and Alexa Fluor 488 AffiniPure donkey anti-mouse IgG (green) (Jackson ImmunoResearch). All stained cells were examined and photographed using a Leica SP5 confocal fluorescence microscope.
Cell apoptosis was assessed as previously described 23 (link).
Cell apoptosis was assessed as previously described 23 (link).
4
Immunofluorescence Imaging of Neuronal Activation
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Two hours after intraperitoneal injection of CNO, mice with AAV8-hSyn-hM3D(Gq)-mCherry or AAV8-CAG-GFP were perfused transcardially using 4% paraformaldehyde (PFA) in 0.1 m PBS. After that, we dissected the brain and postfixed it in the same fixative overnight at 4°C. Then, 75-μm-thick coronal sections were cut using a vibratome (DTK-1500, Dosaka EM Co, Ltd.) from 0.1 to 0.9 mm posterior to the bregma (Franklin and Paxinos, 2008 ). Sections were washed with 0.5% PBS Triton X-100 and incubated in primary antibody against c-Fos (1:500, sc-271243, Santa Cruz Biotechnology) dissolved in 1% blocking reagent in 0.5% PBS Triton X-100 overnight at 4°C. Signal was visualized by a secondary antibody Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500, ab150105, Abcam plc.) or Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (1:500, 715-585-150, Jackson ImmunoResearch) diluted in 0.5% PBS Triton X-100 overnight at 4°C. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml, 422801, BioLegend), mounted with CC/Mount (Diagnostic BioSystems Inc.) and examined under a fluorescent microscope (MVX10, Olympus Corporation) or a laser scanning confocal microscope (FV1000, Olympus Corporation).
5
Pluripotent Stem Cell Marker Detection
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Alkaline phosphatase (AP) staining was performed using an Alkaline Phosphatase Staining Kit II (Stemgent, Cambridge, MA, USA; 00‐0055). For detection of pluripotent stem cell marker antigens, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After washing with PBS, cells were incubated in 0.25% Triton X‐100 (Sigma‐Aldrich; X100‐1L) for 10 min at room temperature, and then blocked with 5% sheep or donkey serum for 30 min at room temperature. Cells were then incubated with the following primary antibodies SSEA‐4 (1 : 100; Invitrogen, Carlsbad, CA, USA; MA1‐021X), TRA‐1‐60 (1 : 50; Invitrogen; MA1‐023), Oct‐4 (1 : 100; Invitrogen; PA5‐27438), SOX2 (1 : 100; Invitrogen; PA1‐094X), and Nanog (1 : 100; Invitrogen; 14‐5768‐82), overnight at 4 °C. Cells were then incubated with appropriate secondary antibodies corresponding to the primary antibody at room temperature for 1 h. The secondary antibodies included Alexa Fluor 594 AffiniPure Donkey anti‐mouse IgG (1 : 250; Jackson, Bar Harbor, ME, USA; 715‐585‐150), Alexa Fluor 488 AffiniPure Donkey anti‐rabbit IgG (1 : 250; Jackson; 711‐545‐152) and Alexa Fluor 594 AffiniPure goat anti‐mouse anti‐mouse IgG (1 : 250; Jackson; 115‐585‐075). The nuclei were stained with DAPI (1 : 10; Vector Laboratories, Burlingame, CA, USA; H‐1200).
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