Cellsense dimensions software

Cells were washed with PBS and fixed with 4% (w/v) paraformaldehyde (Sigma Aldrich) in PBS. Unspecific binding was blocked with 3% (v/v) normal goat serum (NGS, Gibco) and 0.3% Triton-X100 in PBS for 1 h at room temperature. The cells were incubated overnight with primary antibodies (βIII-tubulin, 1:500, Millipore; GFAP, mouse, 1:500, Sigma-Aldrich; Iba-1, 1:500, Wako Chemicals; Olig2, 1:500, Millipore) diluted in blocking solution, washed in PBS, and then treated with anti-mouse-AlexaFluor555 (1:500, goat, Molecular Probes) in blocking solution for 1 h at room-temperature. Cells were washed again with PBS, and DAPI (1:2,000, Sigma-Aldrich) was added to the second-last washing step. The cover slips were mounted on object slides with ProLong® Gold Antifade Mountant (Molecular Probes). A BX61 epifluorescence microscope equipped with CellSense dimensions software (Olympus) was used for image acquisition.

Protein synthesis of Syndecan-1 was shown by immunocytochemistry using a polyclonal antibody against murine Syndecan-1 (CD138) clone 281-2 (1:50, rat anti-mouse, BD Pharmingen) in co-cultured cells after fixation with ice-cold 99% methanol and heparitinase III (2.4mIU/ml, Sigma-Aldrich, H2519-50UN)/ chondroitinase ABC Lyase (0.1U/ml, Sigma-Aldrich, C2905) treatment. Amplification and visualization was performed using secondary antibody (1:50, goat anti-Rat IgG, BD Pharmingen, Heidelberg, Germany) conjugated with biotin (visualised by using Vectastein ALP Kit, AK-5000, Vector Laboratories, Burlinggame, CA, USA) and analysed using a BX51 microscope and cell sense dimensions software (Olympus).

The deformation state of the cell wall of shmooing MATabar1Δ cells was assessed by evaluating the cell shape before and after exposure to high osmotic medium, using a microfluidic device [23 (link)]. The yeast cells were grown to an OD between 0.3 and 0.6 in SD medium and subsequently induced with 10 µM α-factor. After 2 h, cells were seeded into a Y-shaped microfluidic device (MFD). To avoid wash-out during measurement, cells were covalently attached to the glass surface of the MFD (figure 5d) using concanavalin A (Sigma Aldrich). Cell shape was monitored with an IX83 microscope (Olympus Deutschland GmbH, Hamburg, Germany) using a 100× oil immersive objective (Plan S Apo 1.4 NA, Olympus) and a back-illuminated EM-CCD camera (iXON Ultra 888). During the acquisition of z-stack time series, we rapidly changed the extracellular medium from SD to SD with 2 M sorbitol (Sigma Aldrich) using a fluid pressure controller (Fluigent GmbH, Jena, Germany). To ensure constant pheromone induction, we added 10 µM α-factor to both media. Shape was assessed using extended focal images from the Cell Sense Dimensions software (Olympus Deutschland GmbH, Hamburg, Germany).