Anti fade mowiol mounting medium

Example 23

Transfection. HeLa cells were seeded at sub-confluent concentration on glass coverslips (Car Roth, Germany) the day before PEI transfection (Sigma-Aldrich, USA) with 2 μg pENeGFP-PCNA1. After transfection, cells were incubated for 18-24 h at 37° C., 5% CO2. Immunofluorescence. Cells were fixed in 3.7% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 (neoLab Migge Laborbedarf-Vertriebs, Germany) for 10 min, and blocked in 2% bovine serum albumin (Sigma-Aldrich, UK) for 60 min. For GFP labeling, cells were incubated for 60 min with GBP1-Tub-tag-Alexa594 (1:25 or 1:50) prior to extensive washing and DNA counterstain with 1 μg/mL DAPI for 10 min. All steps except fixation were carried out in PBS supplemented with 0.02% Tween 20 (PBST, Carl Roth, 9127.1) at room temperature. Glass coverslips were then mounted with anti-fade Mowiol mounting medium (Sigma-Aldrich, UK).

Confocal Microscopy and Image Analysis. Imaging was carried out with a Leica SP5 II confocal point scanner (Leica Microsystems, Germany) equipped with two HyD hybrid detectors. Image acquisition was performed with a ×60/1.4-0.6 NA Planapochromat oil immersion objective lens. To visualize DAPI, GFP and GBP1-Tub-tag-Alexa594, the 405, 488 and 561 nm excitation lasers were used, respectively. 16 bit images were collected and analyzed with Fiji2.

Example 23

Transfection. HeLa cells were seeded at sub-confluent concentration on glass coverslips (Carl Roth, Germany) the day before PEI transfection (Sigma-Aldrich, USA) with 2 μg pENeGFP-PCNA1. After transfection, cells were incubated for 18-24 h at 37° C., 5% CO2. Immunofluorescence. Cells were fixed in 3.7% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 (neoLab Migge Laborbedarf-Vertriebs, Germany) for 10 min, and blocked in 2% bovine serum albumin (Sigma-Aldrich, UK) for 60 min. For GFP labeling, cells were incubated for 60 min with GBP1-Tub-tag-Alexa594 (1:25 or 1:50) prior to extensive washing and DNA counterstain with 1 μg/mL DAPI for 10 min. All steps except fixation were carried out in PBS supplemented with 0.02% Tween 20 (PBST, Carl Roth, 9127.1) at room temperature. glass coverslips were then mounted with anti-fade Mowiol mounting medium (Sigma-Aldrich, UK).

Confocal Microscopy and Image Analysis. Imaging was carried out with a Leica SP5 II confocal point scanner (Leica Microsystems, Germany) equipped with two HyD hybrid detectors. Image acquisition was performed with a ×60/1.4-0.6 NA Planapochromat oil immersion objective lens. To visualize DAPI, GFP and GBP1-Tub-tag-Alexa594, the 405, 488 and 561 nm excitation lasers were used, respectively. 16 bit images were collected and analyzed with Fiji2.

Example 23

Transfection. HeLa cells were seeded at sub-confluent concentration on glass coverslips (Carl Roth, Germany) the day before PEI transfection (Sigma-Aldrich, USA) with 2 μg pENeGFP-PCNA1. After transfection, cells were incubated for 18-24 h at 37° C., 5% CO2. Immunofluorescence. Cells were fixed in 3.7% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 (neoLab Migge Laborbedarf-Vertriebs, Germany) for 10 min, and blocked in 2% bovine serum albumin (Sigma-Aldrich, UK) for 60 min. For GFP labeling, cells were incubated for 60 min with GBP1-Tub-tag-Alexa594 (1:25 or 1:50) prior to extensive washing and DNA counterstain with 1 μg/mL DAPI for 10 min. All steps except fixation were carried out in PBS supplemented with 0.02% Tween 20 (PBST, Carl Roth, 9127.1) at room temperature. glass coverslips were then mounted with anti-fade Mowiol mounting medium (Sigma-Aldrich, UK).

Confocal Microscopy and Image Analysis. Imaging was carried out with a Leica SP5 II confocal point scanner (Leica Microsystems, Germany) equipped with two HyD hybrid detectors. Image acquisition was performed with a ×60/1.4-0.6 NA Planapochromat oil immersion objective lens. To visualize DAPI, GFP and GBP1-Tub-tag-Alexa594, the 405, 488 and 561 nm excitation lasers were used, respectively. 16 bit images were collected and analyzed with Fiji2.