Donkey anti mouse af647

To achieve the higher labeling density required for dSTORM imaging the staining protocol was slightly modified. Cells were prepared the same way as described up to the storage step in PBS. After storage, cells were blocked with 10% (w/V) BSA (Carl Roth, cat. #8076) in PBS for 30 min, then additionally with Image-iT FX signal enhancer (ThermoFisher Scientific, cat. #I36933) for 60 min. Antibodies and nanobodies were diluted to ~0.5 µg ml⁻¹ in staining/permeabilization solution (PBS, 10% BSA, 0.1% (V/V) Triton X-100 (Sigma-Aldrich, cat. #T8787)). Conventional immunostaining was done at 4 °C for 24 h with the primary antibody (V9, mouse monoclonal, Sigma-Aldrich, cat. #347M-1)^{72 (link)} followed by two washes with PBS, and then stained at 4 °C for 24 h with the secondary antibody (donkey-anti-mouse AF647, ThermoFisher Scientific, cat. #A-31571). For staining with nanobodies, cells were incubated at 4 °C for 48 h. Unbound Nbs were removed by two washes with PBS-T (0.1% w/V Tween-20 (Sigma-Aldrich, cat. #P7949)) and samples were post-fixed with 4% PFA (Sigma-Aldrich, cat. #F8775) and 0.25% (w/V) glutaraldehyde (Sigma-Aldrich, cat. #G5882) in PBS for 5 min to make the binding permanent. Finally, cells were washed twice with PBS to remove fixation solution and stored in PBS with 0.1% (w/V) sodium azide (Carl Roth, cat. #4221) until imaging.

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).

Slides of aortic tissue were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized in 0.1% PBS-Triton for 30 min. The samples were blocked in 5% BSA/PBS/0.1% Triton-X 100 for 1 h and then incubated with the primary antibodies anti-Kruppel-like factor 4 (Klf4, ab214666), anti-neural cell adhesion molecule (Ncam1, ab28486), anti-Ki67 (ab15580), anti-alpha-smooth muscle actin (ab7817) (all from Abcam, USA), and anti-vitronectin (Vtn, MA5-24083, Invitrogen, USA). After several PBS washes, the slides were incubated for 2 h at room temperature with the secondary antibodies AF647 donkey anti-mouse and AF568 donkey anti-rabbit (Invitrogen, USA). Immunofluorescence was assessed using a Zeiss LSM 880 confocal laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany). The fluorescence values were measured with ImageJ software (developed at the National Institutes of Health). At least 100 cells were analyzed for each group.