Tissue tek optimum cutting temperature embedding medium

Alginate beads were fixed with 4% paraformaldehyde (PFA) in Tris-buffered saline (TBS) containing 5mM calcium chloride (CaCl₂) at 4°C overnight and PFA was removed by washing with TBS containing 5 mM CaCl₂. To stabilize bead structure during subsequent manipulations, beads were submerged in polyacrylamide solution (4% acrylamide and 0.13% bis-acrylamide, Bio-Rad Laboratories, Hercules, CA) at room temperature for 2 hours. The polyacrylamide solution was polymerized by adding 1/100 total volume of ammonium persulfate (10% w/v, APS) and 1/1000 total volume of tetramethylethylenediamine (TEMED) into the solution at room temperature for 1 hour. Beads were then submerged in 30% sucrose at 4 °C overnight, embedded in Tissue-Tek optimum cutting temperature embedding medium (Sakura Finetek USA Inc, Torrance, CA), and frozen at −80°C. Frozen blocks of beads were sectioned (60 μm thick) and stored on glass slides. Standard immunofluorescence protocol was conducted to stain Col VI of PCM using a Col VI primary antibody (Abcam, Cambridge, United Kingdom) and secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA).