Orca r2 camera

Cells were then fixed in 10 mM EGTA, 1 mM MgCl2, 20 mM PIPES pH 6.8, 0.2% Triton X-100, and 4% formaldehyde for 10 min, washed 3 times in PBS before incubation in PBS supplemented with 3% BSA for 30 min to block non-specific antibody binding. Next, cells were incubated with primary antibodies for 1 hr, washed 3 times in PBS and then incubated for 30 min with secondary antibodies and DAPI (1:1000 dilution); all antibodies were diluted in PBS + 3% BSA. Cells were then washed in PBS and mounted in Vectashield. For experiments that include Ska staining (i.e., CenpC/Ndc80(C)/Ska3 staining), cells were pre-extracted prior to fixation for 1 min with 10 mM EGTA, 1 mM MgCl2, 20 mM PIPES pH 6.8, 0.2% Triton X-100. Image stacks were acquired using a confocal spinning-disk microscope (VOX UltraView; PerkinElmer, UK) equipped with a 100X / 1.4 NA oil-immersion objective and a Hamamatsu ORCA-R2 camera, controlled by Volocity 6.0 (PerkinElmer) running on a Windows 7 64-bit (Microsoft, Redmond, WA) PC (IBM, New Castle, NY). Image stacks were acquired over 61 z-slices separated by 0.2 μm (for the samples) or over 121 z-slices separated by 0.1μm (for the chromatic shift slide, see below) using the 488, 561, 640 and 405 nm wavelength lasers. Acquisition settings were set in order that the kinetochore signals were typically larger than 50 units above background.

Freshly isolated pyrenoids, obtained from WT cells and in 100 μl BB, were fixed with 4.0% (w/v) paraformaldehyde (BioShop) for 10 min at room temperature. The pyrenoids were then collected by centrifugation at 10,000 x g for 5 min. The pellet was washed twice with BB, resuspended in 100 μl BB, and then immuno-reacted with affinity-purified antibodies against RbcL or RbcS (1:1,000 dilutions, Dr. Spreitzer, University of Nebraska) for 75 min at room temperature with gentle agitation. The conjugates were washed twice with BB, by centrifugation (5,000 x g for 10 min) and resuspension in BB, and finally resuspended in 100 μl BB. This pyrenoid suspension was incubated with Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG secondary antibody (1:200, Invitrogen) for 45 min at room temperature with gentle agitation. The pyrenoid-antibody conjugates were washed again in BB and resuspended in 50 μl BB. Samples were processed as previously described [28 (link)] and observed with a Leica DMI 6000 microscope (Leica) with a 63X/1.4 objective, a Hamamatsu OrcaR2 camera, and Volocity acquisition software (Perkin-Elmer) in the DIC and GFP channel.

H99-GFP strain was streaked from frozen stock on YPD agar and incubated at 30 °C. 2 ml YPD was inoculated with H99-GFP and incubated for ~18 h at 30˚C with rotation. Culture was diluted to OD 0.5 and 100 µL was incubated at 70 °C for 1 h using a thermocycler. One hundred microliters of untreated and heated samples were stained with 10 µg/ml propidium iodide (Invitrogen). Ten microliters of stained samples were loaded onto a hemocytometer and imaged using a ×10 objective and Zeiss AxioImager M2 (×60 Olympus objective) equipped with a Hamamatsu Orca R2 camera and Volocity Software (Perkin Elmer). Images were analyzed using ImageJ/FIJI software. Fluorescence channel images were processed by adjusting the minimum pixel value to 10 and maximum to 90. Number of fluorescent cells for each channel were counted using Measure Particles. Number of double fluorescence positive and double fluorescence negative cells were enumerated manually.