Gtx82661

Immunofluorescence was performed as described previously (Schurmann et al., 2019 ), using 2% (v/v) foetal bovine serum (Life Technologies) in place of normal goat serum. Cells were incubated with primary antibodies against BIN1 (ab54764; Abcam), post-synaptic density 95 (PSD95, D74D3; Cell Signaling), synaptophysin (sc7568; Santa Cruz, TX, USA), glial fibrillary acidic protein (Agilent, ZO334) and microtubule-associated protein 2 (GTX82661; GeneTex) and the appropriate species of AlexaFluor-conjugated secondary antibodies (Life Technologies). Coverslips were mounted onto glass slides using the ProLong Diamond mounting media (Life Technologies). The labelled proteins were imaged under an Eclipse Ti2 inverted Nikon 3D structured illumination microscope, and images were reconstructed using Nikon Imaging Systems Elements software, or a Nikon Eclipse Ti2 inverted microscope with Vt-iSIM scan head and deconvolved using Nikon Imaging Systems Elements software.

Temporal cortex sections (7 μm) of human AD and control brain were prepared from formalin-fixed paraffin-embedded blocks. Sections were deparaffinized in xylene (Thermo Fisher Scientific, X/0250/17) and rehydrated in 99% (v/v) ethanol, followed by antigen retrieval in citrate buffer (0.01 M sodium citrate, pH 6) at 95° to 100°C for 5 min and 65°C for 12 min. Sections were cooled in water and rinsed in tris-buffered saline (TBS), and a hydrophobic wax boarder was applied using PAP pen (SLS, HIS0500). Sections were placed in blocking buffer [1:100 normal goat serum (Sigma-Aldrich, G9023) in TBS] followed by primary antibody incubation overnight: 6E10 (1:200, BioLegend 803002), ALDH1L1 (1:200, Antibodies-online ABIN1304519), CAII (1:100, Abcam ab124687), GFAP (1:500, Dako N1506 or Abcam ab4674), HSPB1 (1:200 Enzo ADI-SPA-803 or HSPB1 mouse Proteintech, 66767-1-Ig), IBA1 (1:200, Wako 019-19741), and MAP2 (1:500, Sigma-Aldrich 05-346 or GeneTex GTX82661). After fluorophore-coupled secondary antibody (Invitrogen) incubation at 1:250, sections were treated with 0.3% (w/v) Sudan Black (Acros Organics, 190160250) in 70% ethanol and mounted using Fluoromount-G mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, 00-4959-52).

Rabbit and rat antibodies to VAPB and PTPIP51 were as described and used at 1:200 for PLAs [10 (link)]. Mouse anti-VDAC1 was from Abcam (ab14734, RRID:AB 443,084) and rabbit anti-IP3 receptor type-1 was from Synaptic Systems (117,003, RRID:AB 2,619,787) and were used for PLAs at 1:200 and 1:100 respectively. Chicken anti-β-Tubulin Isotype III antibody was from Millipore (AB9354, RRID:AB 570,918) and used at 1:500 for immunostaining. Mouse anti-HA epitope-tag (6F2) and mouse anti-Myc epitope tag (9B11) antibodies were from Cell Signaling (H9658, RRID:AB 260,092; 2276 RRID:AB 331,783) and used at 1:500 for immunostaining and 1:1000 for immunoblots. Chicken anti-microtubule-associated protein-2 (MAP2) was from Genetex (GTX82661, RRID:AB 11,172,558) and used for immunostaining at 1:500. Rabbit anti-EGFP was from Invitrogen (A-11,122, RRID:AB 221,569) and used at 1:500. Rabbit anti-serine-9 phosphorylated GSK3β was from Cell Signaling (5558, RRID:AB 10,013,750) and used at 1:100. Species specific goat and donkey anti-mouse, anti-rabbit and anti-chicken Igs coupled to AlexaFluor-488, − 555, -594 or -647 were from Invitrogen and Jackson ImmunoResearch Labs.