No 1.5h

Formalin-fixed, paraffin embedded (FFPE) tissue blocks were generously provided by Prof. Alexander Marx (Pathologisches Institut, UMM/Mannheim Medical Faculty of Heidelberg University, Germany). Anonymous colorectal carcinoma and paired corresponding normal (colorectal) tissue blocks were available from the archival material of the same patients. Approval by the institutional ethics board (Ethics board II at UMM, approval no. 2017-806R-MA) was granted to AM, waiving the need for informed consent for this retrospective and fully anonymised analysis of archival pathological samples.
FFPE blocks were cut using a Leica RM2255 fully automated rotary microtome. The first few sections were discharged and 10 µm thick sections were collected on top of previously cleaned coverslips (No. 1.5H, Paul Marienfeld, Lauda-Könishofen, Germany) from a 37 °C water bath. Coverslips were transferred to a 65 °C oven and left there for a minimum of 30 min. Dried slices were kept at 4 °C for not more than a month before further processing (FISH, confocal microscopy or SMLM).

L4 hermaphrodites were transferred to a new NGM plate with bacteria for 16 h, paralyzed with 50 mM of sodium azide for 5 min, mounted on a freshly prepared 2% (w/v) agarose pad, and covered with an 18 mm × 18 mm coverslip (No. 1.5H, Marienfeld). Imaging was performed on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu) controlled by ZEN 2.3 software (Zeiss). Multiple images covering the entire animal were recorded at 1 × 1 binning with a 40x NA 1.3 Plan-Neofluar objective and assembled into one image using the tiles mode in ZEN.

MP measurements were performed with a TWO^MP (Refeyn Ltd, Oxford, UK) at room temperature (18 °C). Microscope slides (24 × 50 mm, 170 ± 5 µm, No. 1.5H, Paul Marienfeld GmbH & Co. KG, Germany) were cleaned with milli-Q water, isopropanol, milli-Q water and dried with a clean nitrogen stream. Six-well reusable silicone gaskets (CultureWell^TM, 50–3 mm DIA x 1 mm Depth, 3–10 µL, Grace Bio-Labs, Inc., Oregon, USA) were carefully cut and assembled on the cover slide center. After being placed in the mass photometer and before each acquisition, an 18 µL droplet of PBS was put in a well to enable focusing on the glass surface.