Multiplexed Imaging of Alzheimer's Disease Tissue

Using a protocol adapted from Maric et al. [36 (link)] and Murray et al. [37 (link)], paraffin-embedded tissue microarray sections of AD and normal middle temporal gyrus were processed as above. Imaging was carried out with an automated fluorescence microscope (Zeiss Z2 Axioimager) equipped with MetaSystems VSlide slide scanner (MetaSystems) running MetaFer (V 3.12.1) with a 20 × air objective (0.9 NA). This microscope is equipped with 6 custom excitation/dichroic/emission filter sets optimised for spectral separation of compatible fluorophores as previously described (Maric et al. [36 (link)]). Antibodies were then stripped from sections with the addition of 5X NewBlot™ Nitro Stripping Buffer (Li-Cor, NE, USA) for 10 min at room temperature. Sections were then washed in PBS, epitope retrieval performed where necessary, and a subsequent round of immunostaining and imaging performed as above. This was completed over four rounds. Alignment of images from all four rounds was performed using a custom Python script [38 (link)]. We confirmed the effectiveness of stripping at removing previous antibodies in Additional file 1: Figure S5.