Z1 inverted fluorescence microscope

Following each experimental procedure, cells were collected via centrifugation and subjected to two rounds of washing with 1 x PBS. Cells were then fixed and permeabilized using a combination of 4% paraformaldehyde (Sigma-Aldrich/Merck) and 0.1% Triton X-100 (Sigma-Aldrich/Merck). After blocking with 5% BSA (BIO-RAD) in 1 X PBS at room temperature for 1 h, cells were incubated with appropriate primary antibodies at room temperature for 2 h, followed by incubation with species specific Alexa Fluor secondary antibody (Thermo Fisher Scientific Inc.) at room temperature for 1 h. Nuclei were counterstained with 4’, 6’,-diamidino-2-phenylindole (DAPI; BIO-RAD) at room temperature for 30 minutes. Cells were then washed three times with 1 x PBS and mounted using an antifade mounting media (Sigma-Aldrich/Merck). The images were captured using a Zeiss AXIO Observer.Z1 Inverted Fluorescence microscope (Zeiss), and subsequently analysed using ZEN software (Zeiss). The list of primary and secondary antibodies used in immunofluorescence analyses are given in Table 1.

For atomic force microscopy, fibrillar insulin was deposited on freshly cleaved mica and left to settle for 5 min, after which the mica plates were rinsed with milli-Q water and dried under a gentle stream of nitrogen. AFM images were recorded on an NTEGRA Prima setup (NT-MDT, Moscow, Russian Federation) equipped with a gold-coated single crystal silicon cantilever (NSG-01, spring constant ~ 5.1 N/m, resonance frequency of ~ 150 kHz). The images were processed in the Gwyddion software package (Nečas and Klapetek 2012 (link)) using planar subtraction, polynomial background subtraction and correction for linear aberrations. For fluorescence microscopy, 150 µl of insulin solution was placed on positively charged glass (Thermo Scientific, Waltham, MA, USA) and sealed with a 18 × 18 mm coverslip. A Zeiss AxioObserver.Z1 inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) with a 100 × oil immersion objective (NA = 1.46) was used in combination with a Photometrics Evolve EMCCD camera (Photometrics, Tuscon, AZ, USA), 100 ms exposure time, to obtain the images of the samples.

The intracellular localization of the Sec63p-GFP fusion protein was determined by immunofluorescence microscopy. Saccharomyces cerevisiae cells expressing Sec63Δ142-GFP or Sec63Δ237-GFP were grown at 30 °C to 0.4 at an absorbance at 600 nm and untreated (vehicle control) or were treated with 1 μM β-estradiol for 2 h to induce TEV protease expression. The cell cultures were then centrifuged for 5 min at 3000 rpm, washed 2× with sterile distilled water, and resuspended in SD medium (±1 μM β-estradiol) before mounting on slides. Still images of GFP-labeled cells were taken at 63× magnification with a Zeiss Z1 Inverted Fluorescence Microscope using a FITC/GFP filter. In all cases, fluorescent images were focused at the equatorial plane of the cells, and exposure was set to 500 ms.