Alexa series

Cultured SH-SY5Y cells or differentiated SH-SY5Y neurons were briefly washed in cold 1XPBS and fixed immediately with 4% paraformaldehyde in phosphate buffer saline (PBS) for 10 min. The formaldehyde fixed cells were incubated with a permeabilization buffer (0.5% Triton X-100, 100 mM glycine, 1% BSA, 0.7 mM EDTA) for 10 min on ice. The samples were then incubated with a blocking buffer (10% bovine serum, 0.01% sodium azide in 1X PBS) at 37 °C for 1 h or 4 °C overnight. Diluted primary antibodies (TUJ1, Sigma-Aldrich, 1:500; GLP-1R, Santa Cruz, 1:500) were incubated with samples for 1 h at 37 °C. The samples were then rinsed with wash buffer (0.05% Tween-20 in 1x PBS) three times for 5 min at room temperature. A fluorophore conjugated secondary antibody (Alexa series, Invitrogen, Carlsbad, MA, USA; 1:1000 dilution) was added and the samples were incubated for 1 h at 37 °C. Samples were rinsed briefly with wash buffer (3 × 5 min) and mounted with Prolong Gold containing DAPI (nuclear marker, Invitrogen). Images of immunostained neurons were captured using an Olympus FV10i confocal microscope system. The ImageJ (NIH) was used to quantitate the length of differentiated neurites.

IHC was performed as previously described (Ulmke et al., 2021 (link)). Briefly, the antigen retrieval was performed by incubating the brain sections in 0.01 M sodium citrate buffer for 60 min at 70°C, followed by cool-down for 20 min at room temperature. The sections for IHC were then incubated overnight with primary antibody at 4°C after blocking with 5% normal sera of the appropriate species. Incubation with primary antibodies was followed by a 1 h incubation at room temperature with the appropriate A488-, A594-, A555- or A647-labeled (Alexa series, Invitrogen, 1:400) secondary goat or donkey antibodies. Sections were later counterstained with Vectashield mounting medium containing DAPI (Vector laboratories) to label nuclei.