Supplemental Experimental Procedures
gated goat antibodies against rat and mouse IgG were purchased from Mo-lecular Probes. Cells were fixed with –20C methanol fixation for 15 min, postfixed in 4 % paraformaldehyde in PBS for 15 min at room temperature, and rinsed with 1 % Triton X-100 in PBS; subsequent washing and staining steps were...
| Main Authors: | , , , , , , , |
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| Format: | Text |
| Language: | English |
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| Online Access: | http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.530.5370 http://download.cell.com/current-biology/mmcs/journals/0960-9822/piis0960982207024815.mmc1.pdf |
| Summary: | gated goat antibodies against rat and mouse IgG were purchased from Mo-lecular Probes. Cells were fixed with –20C methanol fixation for 15 min, postfixed in 4 % paraformaldehyde in PBS for 15 min at room temperature, and rinsed with 1 % Triton X-100 in PBS; subsequent washing and staining steps were carried out in PBS supplemented with 1 % bovine serum albumin and 0.15 % Tween-20. Immunoprecipitation of the endogenous EB1 was performed with rabbit polyclonal antibodies as described previously [S5]. Image Acquisition and Processing Images of fixed cells were collected with a Leica DMRBE microscope equip-ped with a PL Fluotar 1003 1.3 N.A. oil objective, a FITC/EGFP filter 41012 (Chroma), a Texas Red filter 41004 (Chroma), and an ORCA-ER-1394 CCD camera (Hamamatsu). Twelve-bit images were projected onto the CCD chip at a magnification of 0.1 mm/pixel. Images of fixed samples were pre-pared with Adobe Photoshop by converting them to 8-bit images and linear adjustment of ‘‘levels’’; no image filtering was performed. Simultaneous dual-color (green and red), time-lapse live-cell imaging was performed on the inverted research microscope Nikon Eclipse TE2000E (Ni-kon) with a CFI Apo TIRF 1003 1.49 N.A. oil objective (Nikon), equipped with Table S1. Identification of STIM1 and STIM2 as Potential EB Partners by melanophilin gij87080831 488 551 19.3 23.4 7 8 |
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