Las af

E18 cortical neurons were fixed at DIV 3, 8 or 16, and PIP₃ was detected as described above. All cultures were fixed and labelled using identical conditions. Images were acquired by confocal laser‐scanning microscopy using a Leica TCS SPE confocal microscope. Identical settings were used for each image using Leica LAS AF. Z‐stacks were acquired for each image, spanning the entire depth of either the cell body or the growth cone. PIP₃ fluorescence intensity was measured using Leica LAS AF. Immunofluorescence intensities were calculated by measuring the region of interest (ROI) and subtracting the intensity of a control region adjacent to the ROI.

For statistical analyses, the following software was used: ImageJ, Sigma Plot 2000, and the Leica 3D software suite (LEICA LAS AF). Sigma Plot software was used in to analyze statistically significant differences using Student’s t-test. The Leica 3D software suite (LEICA LAS AF) was used to measure the volume of the beating areas in EBs.

Immunohistochemical preparations were analyzed with a Leica TCS SP5 II confocal laser-scanning microscope equipped with DPSS-, Diode- and Argon-lasers and operated by the Leica Application Suite Advanced Fluorescence software package (LASAF). Images of single frames and maximal projections were compiled using the software LASAF (Leica Microsystems CMS GmbH) and image processing platform Fiji [51 (link)].
Specimens processed for histology were analyzed with a Nikon Eclipse 90i upright microscope and bright-field optics (20x objective).
The precopula was photographed with a Canon 70d camera equipped with an EF-S 18–135 mm f/3.5–5.6 IS objective and a Macro Twin Lite MT-24EX flashlight. Cross-polarized light was used to minimize reflections [52 ].

Neurons were fixed for 10 min using warm 4% PFA (HeLa cells) or 4%/4 %Sucrose (hippocampal neurons). Cells were washed two times with PBS (50 mM NaPO₄, 150 mM NaCl, pH 7.4), permeabilized with ice cold methanol for 8 min at −20 C, and blocked for 1 h in blocking solution (5% goat serum and 1% BSA in PBS). Samples were incubated in primary antibodies diluted in blocking solution for 1 h and were washed three times with PBS; these steps was repeated with secondary antibodies. Following the three PBS washes after secondary antibodies, 0.1 mg/ml of Hoechst 33342 reagent (ThermoFisher, H21492) was added to samples and incubated for 10 min; one last PBS wash occurred after this step. Coverslips were mounted in ProLong Gold (Life Technologies) and images were acquired on a Leica DMI6000B inverted epifluorescence microscope with a 63 × 1.4 NA oil-immersion objective and a Hamamatsu ORCA-R2 charge-coupled device camera driven by LAS-AF (Leica Microsystems) or the previously mentioned Perkin Elmer spinning disk confocal.

MC3T3-E1 cells (cell density of 1 × 10^-3 cells/ml as single cell layer) were seeded on 20 mm × 20 mm glass coverslips (Marienfeld, Germany) and cultured overnight. The cells were then treated with either vehicle (control), E2 (10 nM), GEN (10 nM, 0.1 μM), or ICA (10 nM, 0.1 μM) for 1, 6, and 24 h and/or PI828 (2 μM; 24 h) pre-treatment in steroid-free condition. The anti-apoptotic functions of genstein and icariin in osteoblastic cells under steroid-free culture condition were evaluated at 10 nM and 0.1 μM, concentrations that are likely to be achieved in the human circulation. Treated cells were washed with pre-warmed PBS and incubated with 1 μg/ml AO dye at 37°C for 15 min as described in Hu et al. (2015) (link). AO-stained cells were fixed immediately with 4% paraformaldehyde and mounted on glass slides for scanning. Fluorescence images were captured at mid-plane of cells (oil objective; magnification: 400X) by a Leica TCS SPE DMi8 confocal microscope (Leica Microsystems, Wetzlar, Germany). AO excitation was 458 nm, emission was 480–660 nm (from green to yellow to red fluorescence), indicating different stages of apoptosis (Mpoke and Wolfe, 1997 (link)). The overall intensities of the fluorescent signals of single cell were also quantified using the corresponding Leica Microsystems software station (LAS AF, Leica Microsystems, Germany).

Cells were plated on poly-lysine treated coverslips and subjected to the standard 4-HT treatment to induce Taf1b deletion. At the indicated time points cells were rinsed with PBS, fixed in 4% PFA in PBS for 10 min and permeabilized with 0.5% Triton, PBS for 15 minutes. After a blocking step in PBS-N (PBS, 0.1% IGEPAL (Sigma)), 5% donkey serum, cover slips were incubated with primary antibodies in PBS-N, 5% donkey serum for ~16h at 4 deg. C. RPI was detected using a combination of mouse anti-A194 and A135 antibodies (#SC-293272, #SC-48385), fibrillarin with goat anti-FBL (#LS-C155047), and UBTF with rabbit anti-UBTF (in-house #8). Cells were incubated for ~ 2h at room temperature with the appropriate AlexaFluor or Dylight 488/568/647 conjugated secondary antibodies (ThermoFisher / Jackson ImmunoResearch) and counterstained with DAPI. After mounting in Prolong Diamond (ThermoFisher), epifluorescent 3D image stacks were acquired using a Leica SP5 II scanning confocal microscope and LAS-AF (Leica Microsystems) and Volocity (Quorum Technologies) software.

For embryonic sections, fluorescence signal was detected with an AF6000 Modular Systems Leica fluorescent microscope and documented with a SPOT-CCD camera and the Leica software LAS AF (Version 2.6.0.7266; Leica Microsystems, Wetzlar Germany). For comparison of expression levels, settings were kept constant. For expression and co-localization analysis in the adult murine brain, fluorescence signal was detected using a Zeiss LSM 780 confocal microscope with four lasers (405, 488, 550, and 633 nm) and × 20, × 40, × 63 objective lens. Images were processed using ImageJ.