Alexa 647 labeled secondary antibody

HeLa P4 cells^{82 (link)} were grown on poly-l-lysine coated 12 mm coverslips (No 1.5), permeabilized with 0.003–0.005% digitonin in TPB (20 mM HEPES pH 7.3–7.4, 110 mM KOAc, 2 mM Mg(OAC)₂, 1 mM EGTA, 2 mM DTT and 1 µg ml^–1 each aprotinin, pepstatin and leupeptin). After several stringent washes, nuclear pores were blocked by 15 min incubation with 200 µg ml^–1 wheat germ agglutinin (WGA) on ice. Cells were then incubated for 30 min at room temperature in the absence (background control) or presence of 400 nM recombinant/purified tau protein (K18, K25, hTau40, or Ac-hTau40) or 5 µM TMR-labeled peptides, respectively, in TPB. Tau proteins to be compared quantitatively had similar DOL ( ± 3%) or were corrected to achieve similar DOLs by mixing with unlabeled protein of the same concentration (“DOL corrected”). After several stringent washes to remove non-bound protein, cells were fixed and subjected to immunofluorescence for G3BP1 (Proteintech) and TIA-1 (Santa Cruz, C-20; sc-1751) using Alexa 555 (Thermo Fisher Scientific) and Alexa 647-labeled secondary antibodies (Thermo Fisher Scientific), respectively, to visualize SGs. Tia-1 was stained using an Alexa 647-labeled secondary antibody (Thermo Fisher Scientific), while G3BP1 was stained using either Alexa 488 (with TMR-labeled peptides) or Alexa 555 (with tau proteins) secondary antibodies (Thermo Fisher Scientific).

FFPET sampes were cut into 1.5 μm sections. Samples were deparaffinized in a descending xylene and ethanol series and rehydrated in deionized water for 30 s. Subsequently, antigen retrieval and protein blocking (Dako) were conducted. Anti-mouse CD31 (clone SZ31, Dianova) and anti-mouse CD45 antibodies (clone 30-F11, eBioscience) were applied as primary antibodies. The primary antibody incubation (1 hour) was followed by incubation with Alexa647-labeled secondary antibody (Thermo Fisher) for 30 min in dark. Subsequently, the sections were covered with a DAPI-containing mounting medium (Fluoro-Gel II, Electron Microscopy Sciences, Hatfield, PA, USA). Fluorescence measurements of slides were performed using a slide scanner Pannoramic 250 Flash (3D Histech, Budapest, Hungary). Image visualization was done with the software Pannoramic Viewer (3D Histech, Budapest, Hungary).