Fixed unroofed cells were rinsed with PBS and placed into blocking buffer (PBS with 3 % w/v bovine serum albumin, BSA) for one hour. The unroofed cells were then incubated for one hour in blocking buffer containing 2 μg/mL primary antibody (R-20, Santa Cruz Biotechnology or X22, ThermoScientific). The sample was rinsed with blocking buffer prior to secondary antibody labeling for 30 min (2 μg/mL Alexa Fluor 647 donkey anti-goat, Invitrogen A31571; Alexa Fluor donkey anti-mouse, Invitrogen A21447; or Atto 488 donkey anti-mouse) in blocking buffer. Atto 488 donkey anti-mouse was created with Atto 488 NHS ester (Sigma, 4-molar excess) and unlabeled donkey anti-mouse IgG (Abcam ab6707) and purified through a Superdex 75 10/300 GL size exclusion column (GE Healthcare). Finally, the sample was washed with blocking buffer, PBS, and post-fixed for 20 min. For immunolabeling intact cells, 0.2% Triton X was added to the blocking buffer, and cells were incubated for two minutes in permeabilization buffer (PBS 3 % w/v BSA, 0.5% Triton X) prior to blocking.
Alexa fluor donkey anti mouse
Manufactured by Thermo Fisher Scientific
Alexa Fluor donkey anti-mouse is a secondary antibody used in immunodetection techniques, such as Western blotting and immunofluorescence. It is raised in donkeys and specifically binds to mouse primary antibodies, enabling the detection and visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Atto 488 nhs ester, by Merck Group (2 mentions)
Superdex 75 10 300 gl size exclusion column, by GE Healthcare (2 mentions)
Alexa fluor 647 donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 protocols using alexa fluor donkey anti mouse
1
Unroofed Cell Immunolabeling with Antibodies
2
Immunohistochemical Staining of GFAP
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The cryostat sections were soaked in PBS (0.1 M, pH 7.4), and then incubated in 1% triton PBS solution for 10 minutes in room temperature. Slides were incubated for 1 hour in room temperature in 5% fetal bovine serum solution, and washed in PBS. Then, the sections were soaked overnight at 4°C in a moist chamber with primary antibody to GFAP (Chemicon, Temecula, CA) at 1:400 dilution in PBS 0.1 M+3% serum+0.1% TritonX-100 at 1:100. For secondary antibody, we used Alexa Fluor donkey anti-mouse (Invitrogen; Thermo Fisher Scientific, Waltham, MA) diluted to 1:500.
3
Unroofed Cell Immunolabeling with Antibodies
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Fixed unroofed cells were rinsed with PBS and placed into blocking buffer (PBS with 3 % w/v bovine serum albumin, BSA) for one hour. The unroofed cells were then incubated for one hour in blocking buffer containing 2 μg/mL primary antibody (R-20, Santa Cruz Biotechnology or X22, ThermoScientific). The sample was rinsed with blocking buffer prior to secondary antibody labeling for 30 min (2 μg/mL Alexa Fluor 647 donkey anti-goat, Invitrogen A31571; Alexa Fluor donkey anti-mouse, Invitrogen A21447; or Atto 488 donkey anti-mouse) in blocking buffer. Atto 488 donkey anti-mouse was created with Atto 488 NHS ester (Sigma, 4-molar excess) and unlabeled donkey anti-mouse IgG (Abcam ab6707) and purified through a Superdex 75 10/300 GL size exclusion column (GE Healthcare). Finally, the sample was washed with blocking buffer, PBS, and post-fixed for 20 min. For immunolabeling intact cells, 0.2% Triton X was added to the blocking buffer, and cells were incubated for two minutes in permeabilization buffer (PBS 3 % w/v BSA, 0.5% Triton X) prior to blocking.
4
Immunofluorescence Staining of Cells
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Cells were washed once with PBS and fixed in 4% formaldehyde at room temperature. Subsequently, cells were blocked with 10% donkey serum and 0.1% Triton X-100 in PBS for 1 h, and then diluted primary antibodies were added and incubated at 4°C overnight. After three consecutive washes in wash buffer, cells were incubated for 45 min with secondary antibodies (Alexa Fluor Donkey-anti-mouse and Alexa Fluor Donkey-anti-rabbit, Invitrogen) together with Hoechst 33342 (Invitrogen). After washed, samples were covered with VECTASHIELD mounting medium (Vector) and imaged in Leica SP5 confocal. Acquisition parameter was same for each experiment. Around 100 randomly selected cells were analyzed.
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