8× expansion protocol for the Drosophila CNS

Day 1: Anchoring

• Start with antibody-labeled brains. It is important that brains be permeabilized (e.g. during the antibody staining protocol) before ExM processing.
• Step1: dilute AcX stock solution (10mg/mL) 1:100 in PBS
• Step2: incubate specimens in anchoring solution overnight at RT (500uL in each well for 24-well-plate)
  • Note: use anchoring solution within 15 mins of preparation as AcX hydrolyzes with a half-life ~1 hr
  • Note: cover specimen with foil paper to avoid photo bleaching.
• Step3: prepare gelation chamber, affix silicone gasket to glass slide (use uncharged glass; + charged glass sticks the specimens too strongly).
• Step4: treat gelation chamber/slide with PPL solution, 3 times (one overnight, leave in the dark)

Day2: Gelation 1 (first embedding)

• Step1: wash specimens with 1X PBS
• Step2: mount specimens in gelation chamber and make sure specimens stick down to the PPL treated surface
• Step3: make 8x gelation solution 1 (840ul 8X monomer solution, add 100uL 10XPBS, 20uL 4HT, 20uL TEMED, and 20uL APS). Incubate samples in 8X gelation solution 1 on ice 3X10mins. After the third time, put a cover glass over the gasket, and press gently to seal the edges, then move to 37⁰C for 1-2 hr.
  • Note: prepare 8X gelation solution on ice just before gelling step. Add APS last, no more than 5 minutes before use.
  • Note: be careful not to introduce too many air bubbles as you seal the gasket. Make sure every brain is at least 2mm away from any air bubble or silicone gasket surface, as these inhibit the polymerization process.
• Step4: carefully remove coverslips, trim gels close to the specimens (trim into a right trapezoid shape to keep track of orientation), and recover gels into 12-wells
• Step5: incubate gels in Prok buffer with proteinase K (dilute proteinase K into ProK buffer 1:100). Apply at least ~1mL to each specimen and digest overnight at RT, with shaking

Day3: Gelation2 (second embedding)

• Step1: wash out ProK with PBS 2X30mins
• Step2: equilibrate gels in 10XPBS for at least 15mins
• Step3: measure size of gels by weighing, record weight rounded to the nearest 10mg, and add 10XPBS to each gel up to a total of 100mg (PBS is 1mg/uL).
  • Example: if gel is ~40mg, add 60uL 10xPBS.
  • Note: if gel > 100mg, scale up volumes in this step and the volume of 8x gelation solution 2.
  • Note:if you know the thickness of your gels, you can also get the rough volume by measuring the length and width with a ruler. Gel density is 1mg/uL and 1uL = 1mm^3.
• Step4: make 8x gelation solution 2 (840uL 8X monomer solution, add 20uL 4HT, 20uL TEMED, and 20uL APS on ice as with 8x gelation solution 1). Incubate samples in 8X gelation solution2 on ice with shaking for 40mins.
  • Note: the total volume is 1mL: 100uL of gel + 10xPBS and 900uL of 8x gelation solution 2.
• Step5: make gel sandwich and move it to 37^oC to gel for 2hr.
  • gel sandwich: place each gel onto a glass slide, and put 4 dabs of silicone grease in a 2cm-square pattern around each gel. Place a 22mm-square coverslip on top and press down until the coverslip uniformly contacts the gel. You can press a bit and see that the gel deforms reversibly under gentle pressure. The silicone grease should hold the coverslip in place. Use a pipette to fill the chamber from the side with gelation solution.
• Step6: gently remove the coverslip and trim the second gel. Trim inside the boundary of the first gel so that the entire specimen has both first and second gel.
• Step7: recover gels in PBS, then follow with DAPI stain for 2 hr at RT
• Step8: wash with 1xPBS 2X10mins and leave in PBS at 4^oC until imaging

Solution preparation:

• AcX stock solution (10mg/mL)
  • prepare with DMSO
  • add 500ul DMSO in AcX (Thermo Fisher cat#A20770, 50mg)
  • aliquot into 20uL volumes
  • store at –20^oC with dry beads
• PLL solution
  • Start with poly-L-lysine solution (Ted Pella, cat#18026)
  • Add photo-flo to poly-L-lysine [1:500]. 
  • Store at 4^oC in the dark for up to several months.
• 8X monomer solution
  • Note: Bisacrylamide (mg/mL): 10mg/mL=1%. We dilute the 2% stock to 1% before use because the 2% solution has a strong physical interaction with the pipette tips that makes small volume pipetting prone to errors.

• 0.5 wt% 4HT
  • Weigh 50mg 4HT and prepare in 9.95 mL H₂O
  • aliquot and store at -20^oC
• 10 vol% TEMED
  • 1mL in 9mL H₂O
  • aliquot and store at -20^oC
• 10 wt% APS
  • weigh 1000mg in 9mL H₂O
  • aliquot and store at -20^oC
• ProK buffer