No signal in WB.
Sample: primary rat astrocytes
Cells were lysed in ExB lysis buffer (150 mM NaCl, 20 mM MgCl2, 20 mM CaCl2, 100 mM HEPES, 1% TritonX-100, complete protease inhibitor). 2.7 μg of each protein sample was loaded on a 4–12% polyacrylamide gel (Genescript) and subsequently transferred to nitrocellulose membrane. Membranes were blocked in 5% skimmed milk, 0.01 M Tris–HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.6 in buffer solution at room temperature (RT). Primary antibodies (1:1000) were applied overnight at 4 °C. On the following day, membranes were incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at RT. Specific band size was detected with ECL (GE Healthcare, Brøndby, Denmark) according to the manufacturer’s recommendations and visualized using LAS 4000 (Fujifilm).
Cells: rat astrocytes
cultured for 4 week after isolation
and differentiated with cAMP and hydrocortisone treatment for 1 day
Fixation: 4% paraformaldehyde
Permeabilization: 0.2% triton X-100 in PBS for 10 min
Blocking: 2% bovine serum albumin (BSA) in PBS for 20 min
Primary antibody: 0.002 mg/mL in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Secondary antibody: 1:1000 donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen; A21206) in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Evaluation: strong back grand
On October 4, 2019 Andrea E Toth wrote:
No signal in WB.
Sample: primary rat astrocytes
Cells were lysed in ExB lysis buffer (150 mM NaCl, 20 mM MgCl2, 20 mM CaCl2, 100 mM HEPES, 1% TritonX-100, complete protease inhibitor). 2.7 μg of each protein sample was loaded on a 4–12% polyacrylamide gel (Genescript) and subsequently transferred to nitrocellulose membrane. Membranes were blocked in 5% skimmed milk, 0.01 M Tris–HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.6 in buffer solution at room temperature (RT). Primary antibodies (1:1000) were applied overnight at 4 °C. On the following day, membranes were incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at RT. Specific band size was detected with ECL (GE Healthcare, Brøndby, Denmark) according to the manufacturer’s recommendations and visualized using LAS 4000 (Fujifilm).
On July 4, 2019 Andrea E Toth wrote:
Cells: rat astrocytes
cultured for 4 week after isolation
and differentiated with cAMP and hydrocortisone treatment for 1 day
Fixation: 4% paraformaldehyde
Permeabilization: 0.2% triton X-100 in PBS for 10 min
Blocking: 2% bovine serum albumin (BSA) in PBS for 20 min
Primary antibody: 0.002 mg/mL in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Secondary antibody: 1:1000 donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen; A21206) in 2% BSA, 0.05% triton X-100 in PBS 1h RT
Evaluation: strong back grand