Vectashield4 6 diamidino 2 phenylindole dapi mounting medium

Materials used in generation of organoids were previously described in details^{33 (link)}. The following reagents were used for preparing the organoids for synapse analysis:
Tissue-Tek® O.C.T. Compound (Cat # 25608-930; Sakura, Alphen aan den Rijn, The
Netherlands), Tissue Path™ Disposable Base Molds (Cat # 22-363-553; Fisher Scientific,
Hampton, NH, USA), Goat serum (Cat # 16210064; Invitrogen, Carlsbad, CA, USA), Vectashield
4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Cat # H-1200; Vector Labs,
Burlingame, CA, USA), Adhesive glass slides (Cat # I6172PLUS, Thermo Fisher, Waltham, MA,
USA), Coverslips (Cat # 102222; Thermo Fisher), Kimwipes (Cat # 06-666; Fisher
Scientific), and paraformaldehyde stock (32%) solution (Cat # 15714; Electron Microscopy
Sciences (EMS), Hatfield, PA, USA).
Paraformaldehyde solution for tissue fixation (4% paraformaldehyde solution, pH 7.4) can
be prepared by 1:8 dilution of the 32% stock solution in PBS pH 7.4; 4% paraformaldehyde
solution is to be stored in amber-colored or light-proof containers at 4°C for 1–2
weeks.
The following solutions were used: Blocking Solution (5–10% Goat serum, 0.1% BSA, 0.3%
Triton X-100 in PBS), and Antibody Vehicle Solution (AVS) (1–2% Goat serum, 0.1% Triton
X-100 in PBS).

Cells were seeded at a density of 2.5×10⁵ cells/mL in 1 mL medium on coated glass coverslips and grown overnight. For the primary monocytes, 250 µg/mL fibronectin was used, and for the MM6 cell line, 100 µg/mL collagen was used. The cells were incubated with 500 ng/mL nanoparticles for 4 hours. Subsequently, the coverslips were washed twice with PBS, fixated with 4% paraformaldehyde in PBS for 15 minutes at 37°C, and mounted with a non-hardening 1:20 mixture of Vectashield 4′,6-diamidino-2-phenylindole (DAPI) mounting medium to Vectashield mounting medium (Vector Labs, Burlingame, CA, USA). Localization of nanoparticles was analyzed on a DM IRE2 confocal microscope (Leica Microsystems, Wetzlar, Germany) with a HCX PL APO ×63 oil-immersion objective (Leica Microsystems) and Type F immersion liquid (Leica Microsystems). For excitation, lasers with wavelengths of 405 (DAPI and mTHPP) and 594 nm (SPIONs) were used. The emission was detected between 451 and 471 nm (DAPI), 640 and 660 nm (mTHPP), or 603 and 623 nm (SPIONs).