Axio observer 7.0 apotome 2 0 microscope

For immuno uorescence 25,000 cells of N9 microglia were seeded on 12 mm coverslip (Bluestar) in 24 well plate supplemented with 10% FBS and 1% penicillin-streptomycin. During treatment of hTau40 and ALA the cells were supplemented with 0.5% serum-deprived RPMI media. The treatment was given for 24 hours. Cells were then xed with chilled absolute distilled methanol for 20 minutes at -20°C then washed with 1X PBS thrice. Permeabilisation before staining was carried out using 0.2% Triton X-100 for 15 minutes followed by washing three times with 1X PBS and blocking with 2% serum in 1X PBS for 1 hour at room temperature. Primary antibody treatment was given to cells overnight at 4°C in 2% serum in 1X PBS in a moist chamber. The next day, cells were washed with PBS thrice. Then incubated with secondary antibody in 2% serum at 37°C for 1 hour. Further cells were washed with 1X PBS 3 times and counterstained with DAPI (300 nM). Mounting of coverslip was done in mounting media (80% glycerol). Images were observed under a 63x oil immersion lens in Axio observer 7.0 Apotome 2.0 Zeiss microscope.

N9 cells were passaged in RPMI media supplemented with 10% FBS and 1X penicillin-streptomycin. For immunofluorescence studies, 25,000 cells were seeded on 12 mm coverslip (Bluestar) in 24 well plate. Supplemented with 0.5% serum-deprived RPMI media for the desired treatment. The treatment was given for 24 hours. Cells were then fixed with chilled absolute distilled methanol for 20 minutes at -20°C then washed with 1X PBS thrice. Permeabilisation was carried out using 0.2% Triton X-100 for 15 Minutes, washed three times with 1X PBS followed by blocking with 2% serum in 1X PBS for 1 hour at room temperature. Primary antibody treatment was given to cells overnight at 4°C in 2% serum in 1X PBS in a moist chamber. The next day, cells were washed with PBS thrice. Then incubated in the desired secondary antibody in 2% serum at 37°C for 1 hour. Further cells were washed with 1X PBS 3 times and counterstained with DAPI (300 nM). Mounting of coverslip was done in mounting media (80% glycerol).
Images were observed under a 63x oil immersion lens in Axio observer 7.0 Apotome 2.0 Zeiss microscope.

N9 cells were passaged in RPMI media supplemented with 10% FBS and 1% penicillin-streptomycin. For immuno uorescence studies, 25,000 cells were seeded on 12 mm coverslip (Bluestar) in 24 well plate. Supplemented with 0.5% serum-deprived RPMI media for the desired treatment. The treatment was given for 24 hours. Cells were then xed with chilled absolute distilled methanol for 20 minutes at -20 °C then washed with 1X PBS thrice. Permeabilisation was carried out using 0.2% Triton X-100 for 15 Minutes, washed three times with 1X PBS followed by blocking with 2% serum in 1X PBS for 1 hour at room temperature. Primary antibody treatment was given to cells overnight at 4 °C in 2% serum in 1X PBS in a moist chamber. The next day, cells were washed with PBS thrice. Then incubated in the desired secondary antibody in 2% serum at 37 °C for 1 hour. Further cells were washed with 1X PBS 3 times and counterstained with DAPI (300 nM). Mounting of coverslip was done in mounting media (80% glycerol).
Images were observed under a 63x oil immersion lens in Axio observer 7.0 Apotome 2.0 Zeiss microscope.