Plan neofluar 100 1.30 oil objective

Cells adherent to Lab-Tek chamber slides (177,380) were rinsed in PBS. The cells were treated with BRB80 buffer for 5 min and then fixed for 10 min with 3.7% Formaldehyde in BRB80 buffer. After washing the cells in PBS for 5 min, they were treated with PBST (PBS+0.1% Triton X-100) for 15 min. Finally, the cells were blocked for 20 min in PBST containing 3% BSA. Both control and Tum/RacGAP dsRNA-treated cells were stained with anti-Tubulin diluted 1:200 (S9026 Sigma T clone DM1α), anti-Pav/kinesin-6 diluted 1:200 (kindly provided by D. Glover) or anti-Feo diluted 1:100 (Feo, kindly provided by M. Gatti) antibodies. Immunostained preparations were analysed with a Zeiss Axioskop 2 plus fluorescent microscope equipped with an AxioCam HRm CCD camera and images were acquired with a Plan-NEOFLUAR × 100 1.30 oil objective and Axiovision 4.6.3 software (Zeiss). Photoshop CC and Illustrator CC (Adobe) software were used to prepare the images. To confirm that the Pav/kinesin-6 signal observed in control and Tum/RacGAP RNAi-treated cells was specific to Pav/kinesin-6 primary antibody and not the result of non-specific binding or auto-fluorescence of the secondary antibody, we stained the cells with the Alexa Fluor 594 goat anti-rabbit secondary antibody alone.

Raman spectra from a petrographic thin section were acquired using a WITec Alpha 300 series Raman equipped with a 532 nm laser, operated at 0.53 mW. The laser was focussed with a Zeiss Plan-Neofluar 100×/1.30 Oil objective using Zeiss Immersol 518 F immersion oil. The region of interest was mapped by raster motion in X and Y increments of 0.25 μm with a dwell time of 12 s. To generate mineral maps, Classical Least Squares (CLS) methods used each spectrum as a linear combination of individual constituent spectra, plus error. Using this method, CLS can decompose a spectrum into a set of constituent scores of individual components to produce semi-quantitative mineral maps. Reference spectra of goethite and lepidocrocite for CLS calculations were extracted from within the data set (Supplementary Table 1)^{11 (link)}. The internal standard spectra and representative spectra of goethite and lepidocrocite are provided in Supplementary Figs. 2–3, respectively. To limit the interference by broad fluorescence, the background was removed from all spectra prior to analysis. The baseline was removed using the rolling circle filter with a nominal diameter of 250 as implemented in the “shape” function of the Witec Project 4.1 software^{12 (link)}. Data analysis, including CLS calculations, and instrument control were performed using Project Four software and WITec Control Four, respectively.