Double immunofluorescence was initiated with treatment of a batch of sections from transgenic mouse, monkey, and human cortex in PBS containing 5% donkey serum for 30 min. The sections were then incubated overnight at 4 °C with (1) mouse anti-Aβ antibody 6E10 (1:4000) and the rabbit anti-sortilin antibody (ab16640, 1:1000) or (2) 6E10 (1:4000) and rabbit anti-BACE1. Sections were then incubated at room temperature for 2 h with Alexa Fluor® 488-conjugated donkey anti-mouse IgG and Alexa Fluor® 594-conjugated donkey anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The monkey and human brain sections were treated with 0.1% Sudan Black to block autofluorescence after immunolabeling. All sections were counterstained with bisbenzimide (Hoechst 33342, 1:50,000, catalog number B2261, Sigma-Aldrich, St. Louis, MO, USA) and mounted with antifade medium before microscopic examination.
Alexa fluor 488 conjugated donkey anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Alexa Fluor 488-conjugated donkey anti-mouse IgG is a secondary antibody reagent. It is designed to detect and visualize mouse immunoglobulin G (IgG) in various immunoassay applications. The antibody is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence upon excitation.
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Lab products found in correlation
Alexa fluor 594 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (4 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Bisbenzimide hoechst 33342, by Merck Group (1 mentions)
Ix70 microscope, by Olympus (1 mentions)
Hrp conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Leica confocal microscope, by Leica camera (1 mentions)
Goat anti kim 1, by R&D Systems (1 mentions)
Mouse anti cd44, by R&D Systems (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugated donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence ecl kit, by Beyotime (1 mentions)
Glial fibrillary acidic protein (gfap), by Proteintech (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Lsm 800, by Zeiss (1 mentions)
16 protocols using alexa fluor 488 conjugated donkey anti mouse igg
1
Dual Immunofluorescence Analysis of Amyloid-Beta and Sortilin/BACE1 in Transgenic Mouse, Monkey, and Human Cortex
2
Immunostaining of Myelinated Neurites in DRG Cultures
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After removal of the culture medium, the DRG cultures were washed with phosphate buffered salt solution (PBS) and fixed in 4% paraformaldehyde for 15 min and then washed again with PBS. The fixed cells were permeabilized with 0.1% of Triton X-100 in PBS and then immunoblocked (to avoid nonspecific staining) with a 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The specimens were then double incubated with mouse antimyelin basic protein antibodies (MBP, Covance, Nr. SMI 94 R, 1: 250) to visualize the myelin and rabbit antineurofilament antibodies (NF, Novus Biologicals, 1: 500) to visualize the neurite outgrowth. The primary antibodies were diluted in 0.1% BSA and 0.05% Tween-20 in PBS (diluents buffer) and incubated with the specimens overnight at 4°C. After rinsing with 0.05% Tween-20 in PBS (wash buffer), the DRG specimens were incubated for 1 h at room temperature with the appropriate secondary antibodies: Alexa-Fluor-488-conjugated donkey anti-mouse IgG or Alexa-Fluor-594-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, USA, 1: 800 in a diluent buffer). Finally, the samples were rinsed again with wash buffer and mounted with mounting medium (Immco Diagnostics, USA). Myelinated cultures were alternatively stained with Luxol Fast Blue. All of the images were observed with an Olympus IX70 microscope.
3
Breast Cancer Cell Line Characterization
4
Renal KIM-1 and CD44 Expression
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To examine the expression and distribution of KIM-1 and CD44, 5-µm cryostat kidney sections were incubated with one or two primary antibodies overnight: goat anti-KIM-1 (1:100; R&D Systems, Minneapolis, MN, USA) and/or mouse anti-CD44 (1:100; R&D Systems). The secondary antibodies were Alexa Fluor 488-conjugated donkey anti mouse IgG (1:200) or Alexa Fluor 555-conjugated donkey anti-goat IgG (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). BODIPY 493/503 staining was also performed to evaluate FA accumulation in renal tubules. As a negative control, the sections were exposed to nonimmune IgG (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining was observed. After nuclear staining with DAPI, the slides were mounted with ProLong gold antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). The sections were observed and imaged by a Leica confocal microscope (Wetzlar, Germany).
5
Immunofluorescence and Immunoblotting Analyses
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2,5-HD (purity > 99%) and Hoechst 33342 were purchased from Sigma-Aldrich Co. LLC. (St. Louis, Missouri, USA). Rabbit polyclonal anti-NGF, anti-MAP2, anti-Akt, anti-p-Akt (ser 473), anti-p-Bad (ser 136) and mouse polyclonal anti-Bad were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal anti-Bcl-xL, mouse polyclonal anti-cytochrome c and VDAC were purchased from Abcam Inc. (Cambridge, MA, USA). Goat polyclonal anti-Choline O-acetyltransferase (ChAT) was purchased from Millipore, (Bedford, MA, USA). Mouse monoclonal anti-NGF was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa-Fluor 594 conjugated donkey anti-rabbit IgG, Alexa-Fluor 594 conjugated donkey anti-goat IgG, Alexa-Fluor 488 conjugated goat anti-mouse IgG and Alexa-Fluor 488 conjugated donkey anti-mouse IgG were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Mouse polyclonal anti-β-actin, goat-anti-rabbit horseradish peroxidase (HRP)-conjugated IgG and goat-anti-mouse horseradish peroxidase (HRP)-conjugated IgG were purchased from ZSGB Biotechnology, Inc. (Beijing, China). RIPA lysis buffer, BCA Protein assay Kit, ECL enhanced chemiluminescence kit were purchased from Beyotime Biotechnology, Inc. (Shanghai, China). All other chemicals were of the highest grade commercially available.
6
Immunofluorescence Labeling of Brain Markers
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Immunofluorescence labeling was performed using previously established procedures.18 , 19 The frozen sections were incubated with a mixture of the following primary antibodies overnight at 4°C: STK24 (rabbit, polyclonal; 1:50, bs‐7599R; BIOSS, China), MAP‐2 (guinea pig, polyclonal; 1:200, 188004; SYSY, Germany), and GFAP (mouse, monoclonal; 1:100, 60190‐1‐Ig; Proteintech, China).
On the next day, the sections were incubated with a mixture of corresponding secondary antibodies at room temperature for 1 hour: Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (1:50; 115‐545‐003; Jackson ImmunoResearch, USA), Alexa Fluor 405‐conjugated donkey anti‐guinea pig IgG (1:50; 106‐475‐003; Jackson ImmunoResearch, USA), and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (1:50; 111‐585‐003; Jackson ImmunoResearch, USA) shielded from light for 60 minutes at 37°C. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Japan).
On the next day, the sections were incubated with a mixture of corresponding secondary antibodies at room temperature for 1 hour: Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (1:50; 115‐545‐003; Jackson ImmunoResearch, USA), Alexa Fluor 405‐conjugated donkey anti‐guinea pig IgG (1:50; 106‐475‐003; Jackson ImmunoResearch, USA), and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (1:50; 111‐585‐003; Jackson ImmunoResearch, USA) shielded from light for 60 minutes at 37°C. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Japan).
7
Immunofluorescence Staining of RPE Cells
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After fixation with 4% paraformaldehyde in PBS for 15 min, cells were permeabilized using 0.25% Triton X-100 in PBS for 5–10 min and were blocked for 60 min in 5% goat serum. Then, the cells were incubated for 60 min at room temperature with the following primary antibodies: anti-MITF antibody (Sangon, Cat# D120988, 1:100), anti-ZO-1 antibody (Thermo Fisher Scientific Cat# 40-2200, RRID:AB_2533456 , 1:200), anti-Bestrophin antibody (Novus Cat# NB300-164, RRID:AB_10003019 , 1:100), anti-RPE65 antibody (Abcam, Cat# ab78036, RRID:AB_1566691 , 1:100), anti-PMEL-17 antibody (Abcam, Cat# ab137078, RRID:AB_2732921 , 1:100), Cells were then incubated for 120 min at room temperature with the corresponding secondary antibodies: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat# 711-586-152, RRID:AB_2340622 ), Alexa Fluor 594-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat# 715-586-150, RRID:AB_2340857 ), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat# 711-546-152, RRID:AB_2340619 ) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat# 715-545-150, RRID:AB_2340846 ). Fluorescence images were acquired with a confocal microscope (Zeiss LSM 800, Carl Zeiss).
8
Immunohistochemical Analysis of Alzheimer's Disease
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Free-floating sections were rinsed and blocked with 10% (w/v) normal donkey serum in Tris-buffered saline (TBS) for 1 h at room temperature. Then, the sections were incubated with primary antibodies for 48 h at 4°C. The following primary antibodies were used: mouse anti-Aβ monoclonal antibody (6E10; Covance, 1:500); mouse anti-phospho-tau (ser202, Thr205; AT-8; Thermo Fisher, 1:200); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (Abcam, 1:200); and rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, 1:200). The sections were then rinsed with TBS and incubated for 2 h at room temperature with the respective secondary antibodies, Alexa Fluor-555-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200), Alexa Fluor-488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200) or Alexa Fluor-488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 1:200). After a final rinse in TBS, the sections were mounted on chrome-alum gelatin-coated slides, air-dried and covered with glycerol (diluted in PBS, 1:1, v/v). Staining was visualized using fluorescence microscopy (Leica, Japan; n = 6 per group).
9
Detecting Superoxide and Apoptosis in Neural Stem Cells
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Dihydroethidium (hydroethidine, DHE) staining was performed to detect the production of superoxide anions of cultured NSCs or grafted NSCs. In vitro, NSCs were incubated with 1 μM DHE (Beyotime, China) in a culture medium for 15 min at 37°C. After being washed with PBS, DHE signals were captured with a BX71 fluorescence microscope (Olympus, Japan) and analyzed with Image J software (NIH, USA). In vivo, 200 μl of DHE solution (1 mg/ml, Thermo Fisher Scientific, USA) was intravenously administrated after NSCs transplantation. 2 d after treatment, the mice were killed and brain sections were prepared. DHE and GFP double staining was carried out, followed by labeling with DAPI.
For cultured NSCs, cell apoptosis was assessed by TUNEL-AP (Millipore, USA) staining according to the manufacturer's instructions. For transplanted NSCs, cell apoptosis was detected by one-step TUNEL Apoptosis Assay Kit (Beyotime, China) 2 d after transplantation. Then, the sections were further incubated with anti-GFP (1 : 500, Abcam, UK) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1 : 400, Jackson ImmunoResearch, USA). TUNEL-positive GFP NSCs were counted using unbiased computational stereology as previously described [17 (link)].
For cultured NSCs, cell apoptosis was assessed by TUNEL-AP (Millipore, USA) staining according to the manufacturer's instructions. For transplanted NSCs, cell apoptosis was detected by one-step TUNEL Apoptosis Assay Kit (Beyotime, China) 2 d after transplantation. Then, the sections were further incubated with anti-GFP (1 : 500, Abcam, UK) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1 : 400, Jackson ImmunoResearch, USA). TUNEL-positive GFP NSCs were counted using unbiased computational stereology as previously described [17 (link)].
10
Generating Monoclonal Antibodies against γH2AX
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To generate monoclonal antibodies directed against γH2A.X, serine 139-phosphorylated form of histone H2A.X, mice were immunised with a synthetic peptide 163-6 [CGGKKATQA(phospho-S)QEY] as described previously29 . After generating hybridomas, clones were screened by ELISA using peptides; 163-5 [CGGKKATQASQEY], 163-6 [CGGKKATQA(phospho-S)QEY], 163-7 [CGGKKA(phopho-T)QASQEY], 163-8 [CGGKKA(phopho-T)QA(phospho-S)QEY] and H3S10P [ARTKQTARK(phospho-S)TGGKAPRKQC]. Clone CMA281 specifically reacted with the peptides containing phospho-S139 and isotyped as IgG1-κ using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roch). Antibody purification, Fab preparation and fluorescent dye conjugation were performed as described previously30 . To validate the reactivity of CMA281 to cellular γH2AX in a damage-dependent manner, HeLa cells were untreated or treated with etopside (20 μg/ml; 20 min) and stained with purified IgG followed by Alexa Fluor 488-conjugated donkey anti-mouse IgG (Jackson Immunoresearch) and Alexa Fluor 488-labelled Fab. DNA was counterstained with Hoechst3334230 .
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