Donkey anti mouse af568

Astrocytes isolated from E15 and cultured on poly-D-lysine-coated coverslips were stained with 5 μM MitoTracker DeepRed to label mitochondria. Alternatively, cells were transfected with ER-dsRed expression vector 1 day prior to fixation to label ER. Cells were fixed with 4% paraformaldehyde for 15 min and were then permeabilized by ice-cold methanol for 5 min, followed by PBS rinse for 10 min. The cells were then blocked with 5% bovine serum albumin in PBS with 0.3% TritonX-100 for 1 h, followed by primary antibody [rabbit-anti-HSP60 (CST), rabbit-anti-LC3B (CST), rabbit-anti-LAMP1 (CST), rabbit-anti-ACSL4 (Abcam), mouse-anti-STAT3 (CST), both diluted at 1:200 in the blocking buffer] incubation at 4°C overnight. Then, the cells were incubated in appropriate secondary antibodies [donkey-anti-rabbit-AF488, donkey-anti-rabbit-647, donkey-anti-mouse-488, donkey-anti-mouse-AF568 or donkey-anti-mouse-AF647 (Thermo), all diluted at 1:200 in the blocking buffer] for 1 h at room temperature. After washing three times in TBST, the cells on the coverslip was mounted using Prolong Gold with DAPI (Thermo) and subjected to confocal microscopy observation using the Zeiss LSM880 with Airyscan system. Images were captured using a 63x/1.4NA oil immersion objective. The colocalization analysis was performed with Fiji software using the Coloc 2 plugin.

After fixation and washes in PBS/Triton-X (PBT) 0.5%, brains were blocked overnight in PBS/Triton-X (PBT) 0.5% supplemented with Bovine Serum Albumin (BSA) 1% and then incubated with the following antibodies: α-Imp (rabbit, 1:1000; Medioni et al., 2014 (link)), α-Imp (rat, 1:1000; Medioni et al., 2014 (link)), α-Me31B (rabbit, 1:3000; Lee et al., 2017 (link)), α-Me31B (mouse, 1:3000; Nakamura et al., 2001 (link)) α-pCamkII (rabbit, 1:1000; Santa Cruz Biotechnology, sc-12886-R), α-GFP (chicken, 1:1000; Abcam, #ab13970). After incubation with primary antibodies, brains were washed three times with PBT 0.5% and incubated overnight with secondary antibodies. The following secondary antibodies were used in this study: Goat anti-rat AF568 (Thermofisher, A-11077), Goat anti-rat AF647 (Thermofisher, A-21247) Donkey anti-rabbit AF568 (Thermofisher, A-10042), Donkey anti-rabbit AF647 (Thermofisher, A-31573), Donkey anti-mouse AF488 (Thermofisher, A-21202), Donkey anti-mouse AF568 (Thermofisher, A-10037), Donkey anti-mouse AF647 (Thermofisher, A-31571), and Goat-anti-chicken AF488 (Thermofisher, A-11039). DAPI was used at 5 μg/mL and incubated for 5 min after secondary antibody incubation. Brains were washed in PBT 0.5% three times following secondary antibody incubation and were then mounted in vectashield (Vector Laboratories).