Cells were fixed in 4% paraformaldehyde for 10 minutes, washed with PBS and permeabilized in 0.1% triton in PBS for 10 minutes. Cells were then blocked for 1 hour in 2% serum, 3% BSA and 0.1% triton in PBS and then primary antibody was added in the same solution overnight at 4°C. Cells were washed in PBS for 3 10 minute washes and put in secondary antibody for 1 hour at room temperature. After an additional 3 washes, coverslips were mounted with prolong gold antifade solution (Invitrogen). For surface staining assays, GluA1 antibody was added for 30 minutes to the media live cells at 37°C before fixation. Cells were then blocked without triton and secondary antibody was added immediately following the blocking step. Antibodies used were Brd4 (BethylA301-985A, 1:1000), Arc (Santa Cruz 365736, 1:100), GluA1 (Millipore 2263, 1:300), CK2 (Peirce/Thermo PA5-28686, 1:100), H4K16ac (Abcam 109463, 1:500) and secondary antibodies were AlexaFlour 647 Donkey anti-mouse (Jackson 715-605-150, 1:500) and Rhodamine Red-X goat anti-rabbit (Invitrogen R6394, 1:500).
Alexaflour 647 donkey anti mouse
Manufactured by Jackson ImmunoResearch
AlexaFlour 647 Donkey anti-mouse is a secondary antibody used in immunofluorescence techniques. It is designed to detect and bind to mouse primary antibodies, with the Alexa Fluor 647 fluorescent dye attached to enable visualization under a microscope.
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2 protocols using alexaflour 647 donkey anti mouse
1
Immunofluorescence Staining of Cultured Cells
2
Immunofluorescence Staining of Cultured Cells
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Cells were fixed in 4% paraformaldehyde for 10 minutes, washed with PBS and permeabilized in 0.1% triton in PBS for 10 minutes. Cells were then blocked for 1 hour in 2% serum, 3% BSA and 0.1% triton in PBS and then primary antibody was added in the same solution overnight at 4°C. Cells were washed in PBS for 3 10 minute washes and put in secondary antibody for 1 hour at room temperature. After an additional 3 washes, coverslips were mounted with prolong gold antifade solution (Invitrogen). For surface staining assays, GluA1 antibody was added for 30 minutes to the media live cells at 37°C before fixation. Cells were then blocked without triton and secondary antibody was added immediately following the blocking step. Antibodies used were Brd4 (BethylA301-985A, 1:1000), Arc (Santa Cruz 365736, 1:100), GluA1 (Millipore 2263, 1:300), CK2 (Peirce/Thermo PA5-28686, 1:100), H4K16ac (Abcam 109463, 1:500) and secondary antibodies were AlexaFlour 647 Donkey anti-mouse (Jackson 715-605-150, 1:500) and Rhodamine Red-X goat anti-rabbit (Invitrogen R6394, 1:500).
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