2.5. Study of GFAP expression and confocal imaging

Astrocytes were plated at a density of fifty thousand in 24 well plates (VWR international, 10861–700) and maintained in an incubator. Different concentrations of nicotine (0.1 μM, 1 μM, and 10 μM) were added to the wells and incubated for 24 hours. After 24 hours the media was removed and washed twice by 1X phosphate buffered saline (PBS) solution (VWR international, 45000–446). The cells were then fixed in 4% paraformaldehyde (PFA) in PBS (Thermo Fischer, 101176–014) for 20 minutes and again washed with 1X PBS three times. The cells were then treated with 0.3% Triton-X100 (Sigma-Aldrich) for 15 minutes and washed with 1X PBS two times. Nonspecific staining was blocked by using PBST (PBS solution containing 0.1% of Tween 20 (Thermo Fisher Scientific, 003005) and 10% donkey serum (Sigma-Aldrich, D9663) for 2 hours at room temperature. The cells were incubated with anti-GFAP (rabbit, polyclonal, 1:1000, 2 HR RT, Thermo-Fischer, PA1–10019, RRID: AB_1074611) and then washed with 1X PBS three times. Secondary antibody (anti-rabbit AlexaFluor-488, donkey, polyclonal, 1:1000, Jackson Immuno Research Labs, NC0449336, RRID: AB_2313584) was incubated in the dark for 1 hour. Counterstaining was performed with 0.3 μM of 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (Thermo-Fischer Scientific, D1306) solution (DAPI) in water for 5 minutes. Fluorescence intensity was measured using a fluorimeter. DAPI fluorescence was used to normalize the GFAP fluorescence in each well so that the number of cells would not have any effect on the study of quantitative GFAP expression.

For confocal imaging, astrocytes were cultured in glass bottom dishes, antibody labeling, and fixation was performed as described above. A Nikon A1Rsi laser scanning confocal microscope equipped with a 10X air or 40X air objective was utilized. A 488 nm laser was used for antibody-labelled wild type astrocytes and a 561 nm laser was used for tdTomato-astrocytes.