Ms10uw

Drosophila brains were mounted in glass slides as previously described in Kelly et al. (2017 (link)). The dissected brains were stored in PBS buffer at 4°C in 1.5 mL tubes until ready to be mounted. Then, we glued two #0 coverslips (CG00C2, Thorlabs) on a glass slide (MS10UW, Thorlabs) by using nail polish (Figure 7E). The distance between the two coverslips was around 5 mm. A pipette was used to pick up a brain from Eppendorf tube and put the brain on the glass slide between the two #0 coverslips. Then, the brain was placed in the correct orientation with the help of a 20X magnification (LCAch N 20X, Olympus) stereo microscope (NAME NUMBER). Then, we added 10 μl of 1% agarose diluted in PBS on the brain. Before the agarose became solid, a #1.5 coverslip was mounted on the brain and attached to the #1 coverslips. The left and right sides of the coverslip were sealed with nail polish and the front and backside were sealed by using two-component gel (Figure 7D).

Firstly, we put two strips of double sided tape on a glass slide (MS10UW, Thorlabs) as spacer (Figure 7C). The spheroid Cao2-BBE cell was stored in PBS in Eppendorf tube. We picked up the spheroid Caco2-BBE cell from the Eppendorf tube by pipette and put the cell on a #1.5 coverslip (CG00C2, Thorlabs). Then, 20 μl of 1% agarose (BP160-100, Thermo Fisher Scientific, Waltham MA, U.S.A.) diluted by PBS was added to stabilize the cell. Before the agarose became solid, the coverslip with the cell was mounted on a glass slide attached with the double sided tape. Then, we used nail polish to seal the edge between the coverslip and the double sided tape. After the nail polish was dry, we added the dSTORM buffer into the chamber and used two-component gel (Picodent, Wipperfürth) to seal the front and backside of the coverslip (Figure 7F).

For Lphn2 and synaptic immunofluorescence, Lphn2^mVenus transgenic mice were perfused transcardially with 20 mL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, pH 7.4) and 10 mL fresh 4% paraformaldehyde (PFA) in PBS. Brains were dissected out, placed in 4% PFA at 4°C overnight, briefly rinsed in PBS, and mounted in agarose. Horizontal sections (100 μm) were prepared using a VT1200S vibratome (Leica). Dorsal hippocampal sections were washed for 5 min in PBS and blocked for 1 h at room temperature with 10% normal goat serum (NGS; ab7481, Abcam) and 0.5% Triton X-100 in PBS. Subsequently, slices were incubated overnight at 4°C in PBS containing 1% NGS, 0.01% Triton, and primary antibody. Sections were washed with PBS 3 times for 5 min each and then incubated in PBS containing 1% NGS, 0.01% Triton, and secondary antibody for 4 h at room temperature. Sections were washed again with PBS (3 washes, 5 min each) and then mounted on glass slides (MS10UW, Thorlabs) using Vectashield Plus Antifade mountant (Vector Laboratories, H-1900-10) and #1.5H high-precision cover glass (CG15KH, Thorlabs).