Plan neofluar objective

After culturing the cells were fixed with 4 % PFA in PBS. The actin cytoskeleton was stained with Alexa 488-conjugated phalloidin (200 U/ml stock diluted 1:100 in PBS; Invitrogen Europe, Paisley, UK) for 20 minutes at +37 °C. Nuclei were stained with Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS; Sigma-Aldrich) for 10 minutes at room temperature. Staining for osteoclast-specific enzyme TRACP was carried out with a commercial acid phosphatase leukocyte kit (Sigma-Aldrich) for 20 minutes at +37 °C. The samples were mounted in 70 % glycerol-PBS and viewed in a Nikon Eclipse E600 fluorescence microscope (Tokyo, Japan) and Plan 10 × objective. Multinuclear cells with three or more nuclei were counted from each bone slice from five randomly chosen microscope fields, bone slice n ≥ 3. The number of nuclei were counted from 4 multinuclear cells from 5 randomly chosen areas. Images were taken with QImaging MicroPublisher 5.0 RTV camera and QCapture 2.90.1 software (QImaging, Surrey, Canada). Confocal images were taken with LSM 510 META confocal microscope combined with an Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) with 20 x Plan Neofluar objective (Carl Zeiss).

Excised tissues from fluorescein-dextran (FDA)–, cascade blue–dextran-, or rhodamine-dextran (RDA)–injected embryos were dissociated in Ca²⁺-free MBS, and cells were mixed and filmed (two to three independent repetitions per treatment) while reaggregating in MBS in BSA-coated dishes at room temperature using an Axiovert 200M inverted microscope with a 20× Plan-Neofluar objective, numerical aperture 0.5, an AxioCam MRm, and AxioVision 4.8 image processing software (all Carl Zeiss).

Cells were rinsed in PBS and fixed in 3.7% formaldehyde (v/v in PBS) for 15 min. Fixed cells were washed twice with PBS for 5 min each and permeabilized in 0.3% Triton-X 100 for 10 min. This was followed by incubation in blocking buffer (3% bovine serum albumin in PBS) for 1 h. To detect endogenous PABPN1 and transfected myc-tagged proteins, fixed cells were incubated overnight at 4°C with anti-PABPN1 (1:500), anti-MATR3 (1:1000, Bethyl labs) or anti-myc (1:500, Cell Signaling) antibodies followed by staining with Texas Red or fluorescein-conjugated secondary antibodies (Jackson Labs). Position of the nucleus was marked by incubation with Hoechst 33342 (ThermoFisher Scientific) or DAPI (Sigma). Fluorescent or Phase images were acquired using a microscope (Axiovert 200 M; Carl Zeiss MicroImaging, Inc.) with a 0.3 NA 10× (Cellular Proliferation Assay), 20× (Phase images for differentiation), 60× (Neat1 FISH) or 100× (SFPQ staining) Plan-Neofluar objective (Carl Zeiss MicroImaging, Inc.) and camera (QImaging) with OpenLab 5.5.0 software (Improvision).

Differential interference contrast and fluorescent images were captured with an Axioimager A1 (ZEISS) equipped with epifluorescence (Filter Set 13 for GFP [excitation BP 470/20, beam splitter FT 495, emission BP 503–530] and Filter Set 20 for Cherry [excitation BP 546/12, beam splitter FT 560, emission BP 575–640]) and an AxioCam monochrome digital camera (ZEISS). Images were processed and viewed using Axiovision Rel. 4.7 software (ZEISS). A 100× Plan-Neofluar objective (NA1.30) was used with Immersol 518F oil (ZEISS). Confocal images were captured by an LSM 5 Pascal (ZEISS) inverted confocal microscope with 488-nm (emission filter BP 503–530) and 543-nm (emission filter BP 560–615) lasers, and images were processed and viewed using LSM Image Browser software (ZEISS). All images were taken at 20°C.
To quantify the fluorescence intensity of LMP-1::sfGFP, 15 images were taken of each cell type with equal exposure time and quantified using ImageJ 1.42q (National Institutes of Health).