Va 044 photoinitiator

Polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS) filaments were purchased from 3D FilaPrint (UK); 40% acrylamide and 2% bis-acrylamide solutions from Bio-Rad (UK); 16% paraformaldehyde (PFA) solution from Alfa Aesar (UK); VA-044 photoinitiator from Wako Chemicals (Germany); agarose from Roche (UK); sodium dodecyl sulfate (SDS), boric acid and sodium hydroxide (NaOH) from Sigma–Aldrich (UK); phosphate buffered saline (PBS) tablets and glycerol from Fisher Scientific (UK).

12‐week‐old C57BL/6 mice and Sprague–Dawley rats weighing between 250–300 g were terminally anaesthetized with sodium pentobarbital (intraperitoneal injection; 120 mg/kg body weight) and transcardially perfused with ice‐cold phosphate‐buffered saline (PBS, pH7.4) and then with hydrogel monomer solution consisting of 4% paraformaldehyde (PFA), 2% or 4% (vol/vol) acrylamide (Bio‐Rad, UK), 0.05% (vol/vol) bis‐acrylamide (Bio‐Rad, UK), 0.25% (wt/vol) VA‐044 photoinitiator (Wako, Alpha‐Labs, Eastleigh, UK) and PBS. Brains were then extracted and immersed in the same solution for 2 days at 4°C. Next, the brains with hydrogel solution were transferred into a 50 ml container and a layer of olive oil was poured on top of the solution with the lid tightly screwed to prevent oxygen inhibition of the subsequent polymerization of the hydrogel by incubating at 37°C for 3 h (or 4 h for 2% acrylamide solution). After removal of excess hydrogel from the hydrogel‐tissue hybrid, brain tissues were left either undissected, bisected into two hemispheres in the case for mouse brains or cut into 3‐mm‐thick coronal sections for rat brains using a rat brain matrix before proceeding to the subsequent clarification step.

Neutral-buffered 4% paraformaldehyde solution (NB-PFA, pH = 7.0, Gadot, Israel) was used for tissue fixation. For hydrogel preparation, we used Photoinitiator VA-044 (Wako, USA), Acrylamide 40% solution and Bis- acrylamide 2% solution (Bio-Rad, Israel), Boric Acid, Urea, Glycerol, Triton X-100, Sodium hydroxide (NaOH) pellets and Sodium dodecyl sulfate (SDS) (Sigma, Israel). For immunolabeling the following antibodies were used: rabbit anti mouse LYVE1 antibody (Fitzgerald, USA); donkey anti rabbit Alexa fluor-647 antibody (Abcam, USA) and normal goat serum (NGS, Biological industries, Israel). To induce ovulation, pregnant mare serum gonadotropin (PMSG, National Hormone & Peptide Program, USA) was used.