Anti mfsd5

Proximity ligation assay (PLA) was run on paraffin embedded mouse brain section as previously described [27 (link)]. The following antibody parameters were used: Anti-MFSD5 (1:400, Santa Cruz) vs Anti-MFSD5 (1:50, MyBioSource) and anti-MFSD11 (1:80, Sigma-Aldrich) vs anti-MFSD11 (1:80, Santa Cruz). DAPI was included in all set ups. Images were taken using the Zeiss Axioplan2 fluorescent microscope.

Floating sections were washed in Tris-buffered saline (TBS) followed by incubation in 10% Methanol (Sigma-Aldrich) and 4% hydrogen peroxide (Merck, Germany) diluted in TBS for 10min. After additional washes tissues were blocked for 1h in supermix before the primary antibodies (anti-MFSD5 (1:500, Santa Cruz), anti-MFSD11 (1:500, Sigma-Aldrich)), diluted in supermix were added and allowed to incubate at 4°C overnight. Sections were then washed in TBS and secondary biotinylated antibodies (1:400, Vector laboratories) were added and incubated for 1h at RT. After additional TBS washes, 1h incubation in the ABC kit solution (Vector laboratories) was performed. The samples were then developed in a solution consisting of one DAB tablet (Sigma-Aldrich) dissolved in 12,5ml TBS, 3.8% NiCl and 0.03% hydrogen peroxide added directly prior development for a maximum of 10min, the reactions were stopped by TBS washes. Sections were transferred to gelatine-coated slides (Superfrost, Menzel-Gläser, Thermo Scientific) and dried overnight before dehydrated by an ethanol series completed with Xylene (Sigma-Aldrich) and mounting in DPX (Sigma-Aldrich). Sections stained without primary antibodies were used as negative controls. Samples were scanned in a Pannoramic midi scanner using bregma according to The mouse brain atlas [28 ].