After perfusion, mouse brains were harvested and dissected followed by myelin removal by centrifugation on a 30% Percoll gradient. Brain pellets were sequentially stained with Ghost Dye violet 510 (1:1,000; Tonbo Biosciences) followed by incubation with the microglial surface markers anti-CD11b, anti-CD45, and anti-CD11c for 30 min. Subsequently, stained samples were fixed and permeabilized using Intracellular Fixation & Permeabilization Buffer (eBioscience). Intracellular staining was performed for the presynaptic marker anti-synaptophysin (1:100; Invitrogen) or postsynaptic marker PSD95 (1:100; Invitrogen) followed by staining with Alexa Fluor 488–donkey anti-mouse IgG (H+L) secondary antibody (1:300; Invitrogen). Samples were acquired on a CytoFLEX (Beckman Coulter) flow cytometer followed by analysis with FlowJo v10 (Tree Star).
Alexa fluor 488 donkey anti mouse igg h l secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488–donkey anti-mouse IgG (H+L) secondary antibody is a fluorescently labeled antibody that binds to mouse immunoglobulin (IgG) heavy and light chains. It is designed for use in various immunodetection techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting.
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Lab products found in correlation
Anti synaptophysin, by Thermo Fisher Scientific (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Ghost dye violet 510, by Cytek Biosciences (1 mentions)
Intracellular fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Caffeine, by Nacalai Tesque (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
H2ax ser 139, by Merck Group (1 mentions)
Triton x 100, by Nacalai Tesque (1 mentions)
2 protocols using alexa fluor 488 donkey anti mouse igg h l secondary antibody
1
Multimodal Microglial Profiling in Mouse Brain
2
Immunofluorescence Assay for DNA Damage
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Human RPE cells were grown in MEMα (Wako, Osaka, Japan) supplemented with 10% FBS at 37°C with 5% CO 2 . DNA replication stress was induced by exposure to 1 μM CPT (TopoGEN, Buena Vista, VA, USA) with or without 1 mM caffeine (Nacalai Tesque, Kyoto, Japan).
Immunofluorescence Staining of γH2AX RPE cells were fixed with 4% paraformaldehyde (Nacalai Tesque)/phosphate-buffered saline (PBS) for 10 min. After fixation, samples were washed 3 times with PBS and permeabilized with 0.5% Triton X-100 (Nacalai Tesque)/PBS for 15 min at room temperature (RT). After washing with PBS, blocking was done in 3% Blockace (Yukijirushi, Hokkaido, Japan)/PBS for 1 h at 37°C. Anti-phospho-histone H2A.X (Ser139) (1:500; Merck, Darmstadt, Germany) was the primary antibody, followed by Alexa Fluor ® 488 donkey anti-mouse IgG (H + L) secondary antibody (1:500; Invitrogen, Waltham, MA, USA, cat. #21202). Both primary and secondary antibody reactions were performed at 37°C for 1 h.
Immunofluorescence Staining of γH2AX RPE cells were fixed with 4% paraformaldehyde (Nacalai Tesque)/phosphate-buffered saline (PBS) for 10 min. After fixation, samples were washed 3 times with PBS and permeabilized with 0.5% Triton X-100 (Nacalai Tesque)/PBS for 15 min at room temperature (RT). After washing with PBS, blocking was done in 3% Blockace (Yukijirushi, Hokkaido, Japan)/PBS for 1 h at 37°C. Anti-phospho-histone H2A.X (Ser139) (1:500; Merck, Darmstadt, Germany) was the primary antibody, followed by Alexa Fluor ® 488 donkey anti-mouse IgG (H + L) secondary antibody (1:500; Invitrogen, Waltham, MA, USA, cat. #21202). Both primary and secondary antibody reactions were performed at 37°C for 1 h.
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