Plan apochromat 63x 1.4 numerical aperture oil objective

Sections were washed with PBS and incubated for 30 minutes in blocking buffer (10% Normal Donkey Serum (NDS), 1% BSA, 0.3% Triton in PBS). Afterwards, primary antibodies rabbit anti-FTH1 (D1D4; 4393S, Cell Signaling) and goat anti-Iba1 (Abcam, ab5076) were used at 1:500 and incubated overnight at 4°C in 1% NDS, 1% BSA, 0.3% Triton in PBS. Next day, sections were washed 3× for 10 minutes each with PBS and incubated with Alexa Fluorophore (568 or 647)-conjugated secondary antibodies for 1 hour at room temperature (RT). After 3 washes with PBS, sections were mounted on glass slides, coverslipped using Prolong Gold Antifade mounting media (Invitrogen), and imaged using a Zeiss LSM 880 confocal microscope equipped with Plan-Apochromat 63X/1.4 numerical aperture oil-objective (Carl Zeiss AG). Sections were further imaged using an Olympus VS120 (Olympus, Center Valley, Pennsylvania) slide scanner microscope.

Sections were washed with PBS and incubated for 30 min in blocking buffer (10% Normal Donkey Serum (NDS), 1% BSA, 0.3% Triton in PBS). Afterwards, primary antibodies were used at 1:500 and incubated overnight in 1% NDS, 1% BSA, 0.3% Triton in PBS. Next day, sections were washed three times with PBS and incubated with 1:500 Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. After three washes with PBS, sections were mounted on glass slides, coverslipped using Prolong Gold Antifade mounting media (Invitrogen) and imaged using a Zeiss LSM 880 confocal microscope equipped with Plan-Apochromat 63X/1.4 numerical aperture oil-objective (Carl Zeiss AG).