Alexa 594 anti rabbit igg

For detecting the NE, each 2-μl extract sample was gently mixed on a glass slide with 4 μl of the fixing solution (3.7% formaldehyde, 2 μg/ml Hoechst 33258, 50% glycerol, 80-mM KCl, 15-mM NaCl, 15-mM Pipes-KOH, pH 7.2) containing 10 μM of 3,3′-dihexyloxacarbocyanine iodide (Sigma-Aldrich) and squashed under a 22-mm × 22-mm square coverslip.
For detecting replication activity and immunofluorescence, each 10 μl extract sample was diluted with 90-μl of extraction buffer (EB) (100-mM KCl, 2.5-mM MgCl₂, 50-mM Hepes-KOH, pH 7.5), 11 μl of 37% formaldehyde was then added, incubated at RT for 10 min, 1 ml of EB was added further, which was loaded into a swinging bucket for collecting suspension culture cells (CS-2, Tomy). Nuclei were collected by centrifuge (500g, 5 min) onto poly-L-lysine–coated coverslip (IWAKI) through 0.5-ml of EB plus 30% sucrose layer. DNA replication was labeled with 1 μM of CF594-dUTP (Biotium). Nuclear lamin B1 was detected by sequential incubation with anti-lamin B1 antibody (ab16048, Abcam) and Alexa 594 anti-rabbit IgG (Thermo Fisher). The coverslips were washed with TBS-T, PBS, and dH₂O and mounted on glass slides with 3 μl of SlowFade Gold (Thermo Fisher) containing 2 μg/ml Hoechst 33258.

The animals were subjected to anaesthesia before fixation at stage 40 by 2 h of incubation in methanol 100% at room temperature (RT). Samples were rinsed three times with PTw 1× (1× PBS, 0.1% Tween, pH 7.3) and then incubated overnight in 15% sucrose/PTW1X at 4°C, and then again incubated overnight in 30% sucrose/PTW1X at 4°C. Cryosections of the larvae were processed for immunostaining as follows: rehydrated in 1× PBS for 30 min, washed in PBS‐0.1% Triton X‐100 and treated with antigen retrieval solution [proteinase K 20 mg/ml (Sigma‐Aldrich, Germany) dissolved in 10 mM Tris pH 8.0, 1 mM EDTA (TE)] for 15 min at 37°C. Cryosections were then permeabilized with 0.5% Triton X‐100 in 1× PBS for 20 min at RT, rinsed in PBS 0.1% Triton X‐100 and moved to blocking solution [2% BSA, 2% serum, 2% DMSO in PBS‐0.1% Triton X‐100] for 30 min at RT. Cryosections were incubated with rabbit anti‐collagen type II (Rockland, 1:400) and mouse anti‐LC3B (Nanotools, 1:100) antibodies overnight at 4°C, then washed with PBS‐0.1% Triton X‐100 and incubated with secondary antibodies, Alexa‐594 anti‐rabbit IgG (1:500), Alexa‐488 anti‐mouse IgG (1:500; Thermo Fisher) for 1 h at RT. Nuclei were stained with DAPI (1:500).

Both HBMEC and N9 cells were grown in 24-well plates with coated coverslips and immunostaining performed as already described^{62 , 64 (link)}. Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology). Incubation with secondary antibodies Alexa 594 anti-rabbit IgG (1:500) and Alexa 488 anti-mouse IgG (1:500) (Invitrogen) lasted for 2 h at room temperature. Nuclei were counterstained with DAPI. Between incubations cells were washed three times with PBS. HBMEC staining was examined using a Leica DFC 490 camera (Leica, Germany) adapted to an AxioScope.A1 microscope (Zeiss, Germany), ZEN 2012 blue edition software by Carl Zeiss Microscopy GmbH, 2011. Confocal fluorescent Z-series N9 cells were acquired using on a Leica SP5 live upright confocal (Leica, Wetzlar), using a 63 × 1.3NA oil immersion objective, the UV lamp and DPSS 561 nm yellow-green laser. Post-acquiring treatment was performed using ImageJ software (NIH, USA).