Alexa fluor fluorescent dye conjugated secondary antibodies

Thirty-micrometer-thick sagittal cryosections of mouse brain were cut and mounted on glass slides. The sections were pretreated with 0.01 mol/L citrate buffer (pH = 6.0) for 20 min and then were blocked for 1 h with 10% goat serum in PBST (0.25% triton-X100). Following blocking, the sections were incubated with primary antibodies, overnight at 4 °C. The next day, the sections were washed and incubated with Alexa-Fluor fluorescent dye-conjugated secondary antibodies Invitrogen, USA; 1:500) in PBS for 90 min at room temperature. The primary antibodies were specific to mouse monoclonal human TDP-43 (Abnova, Taiwan; 1:500), mouse polyclonal GFAP (Cell Signaling Technologies, USA; 1:500), rabbit polyclonal Iba1 (Wako Chemicals, USA; 1:500), mouse monoclonal NeuN (Cell Signaling Technologies, USA; 1:1000) Nuclear factor kappa-B (NF-κB) (Santa Cruz;1:1000), phospho-NF-κB (Ser 536) (Cell Signaling Technologies, USA; 1:500), arginase-1 (Santa Cruz; 1:1000), and Ym-1 (Stem Cell Technologies, Canada;1:500) protein markers. Four equidistant sections for the hippocampus and six equidistant sections of the cortex were assessed for every mouse. All sections were imaged using confocal microscopy (LSM microscope 700, Zeiss). The ImageJ software was used for analyzing and quantifying the immunoreactive areas.

Immunostaining of rat primary hippocampal neurons or HT-22 cells employed established protocols (Baumert et al., 2020 (link)). Briefly, cells cultured on poly-D-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 0.5% Triton X-100 solution for 15 min. Following fixation and permeabilization, cells were blocked with PBS containing 1% bovine serum albumin overnight at 4°C. Cells were then incubated with the indicated primary antibodies overnight at 4°C and subsequently rinsed three times with fresh PBS for 10 min each, followed by an overnight rinse with PBS at 4°C. Cells were then incubated with Alexa Fluor fluorescent dye-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature before the coverslips were washed and finally mounted onto glass slides with Vectashield (Vector Laboratories) mounting solution. Cells were visualized using a Nikon T2i Inverted Confocal Microscope with an Apo-Plan 60 × 1.4 NA oil objective at room temperature. Images were captured with a Nikon A1-DUG GaAsP hybrid four-channel multi-detector and Nikon NIS-Elements software. All z-series images were acquired at a pixel size of 100 nm and a step size of 0.2 μm.