Alexa fluor 555 donkey anti mouse igg

Day 1. Free-floating sections (40 μm thick) were placed in 24 well plates, rinsed in PBS-Triton X100 (TX), and blocked for 60 min with blocking buffer (BB, 10% normal goat serum in PBS-TX and 0.05% NaN). Sections were then incubated overnight at 4 °C under slight agitation in a BB solution of two combined primary antibodies: a rabbit anti-Iba1 antibody + a mouse anti-β amyloid antibody.
Day 2. After washing, the sections were incubated for 2 h at room temperature in the dark with AlexaFluor 555 donkey anti-mouse IgG (1:400) secondary antibody diluted in BB and then for 2 h at room temperature in the dark with AlexaFluor 555 donkey anti-mouse IgG (1:400, Code #A31570, Thermo Fisher Scientific, Milan, Italy) + AlexaFluor 635 goat anti-rabbit IgG (1:400, Code #A31577, Thermo Fisher Scientific). After washing, the astrocytes were immunostained using a mouse anti-GFAP antibody conjugated with the fluorochrome AlexaFluor 488 (1:500, Code #MAB3402X, Millipore, Billerica, MA, USA).

Kidney and liver samples were fixed in 4% paraformaldehyde (Merck) and embedded in paraffin. Blocks were cut in 1–4 µm sections, dewaxed, and rehydrated through a series of Tissue-Clear (Sakura ProHosp, Alphen an den Rijn, The Netherlands) and ethanol (99–70%) (Merck). Antigen retrieval was performed with TEG-buffer (10 mM TrisBase, 0.5 mM EGTA Tritiplex VI) (Merck) in a microwave oven for 20 min. Sections were washed in TBST (Merck) and blocked for 30 min in 3% Bovine Serum Albumin (BSA)/TBST (Merck), washed and blocked 10 min in hydrogen peroxide. Anti-MASP-2 (15-17-3) was applied undiluted/in culture supernatant overnight at 4 °C, washed, and incubated with HRP-conjugated goat-anti mouse-IgG (P0447; Dako) diluted 1:1000 for 1 h. Staining was visualized with 3.3′ diaminobenzidine (DAB)(Dako) and counterstained with Mayer's Hematoxylin (Merck). Images were taken with Olympus BX51 microscope with a DP26 camera and Cell Sens software (Olympus, Tokyo, Japan). Co-localization was investigated using anti-MASP-2, anti-AQP2 (collecting duct, principal cell marker, 1:200, 9882, Santa Cruz) and anti-ATP6V1B1 (collecting duct, intercalated cell marker, HPA031847, 1:50), respectively. AlexaFluor488 donkey-anti goat-IgG (Invitrogen) and AlexaFluor555 donkey-anti mouse-IgG (Invitrogen) were used as secondary antibodies.

First, 5 × 10³ cells/chamber HAP1 cells were cultured in an 8-well slide and chamber (192-008, Watson, Tokyo, Japan). Cells were fixed with 4% PFA in PBS (26123-55, Nacalai Tesque), washed with PBS three times, and blocked with blocking buffer (5% skim milk with 0.3% triton X-100). Primary antibodies (anti-TLS/FUS (ab154141, Abcam, Waltham, MA, USA), anti-ZAP3 (A304-038A, Bethyl, Montgomery, TX, USA), anti-Matrin3 (A300-590A, Bethyl)) were diluted with blocking buffer at 1:1000, anti-TIA-1 (ab140595, Abcam) and anti-TDP-43 (12892-1-AP, Proteintech, Rosemont, IL, USA) were diluted at 1:250, and incubated at 4 °C for 16 h. Cells were washed for three times, and incubated with secondary antibodies (AlexaFlour 647 goat anti-rabbit IgG (A21244, Invitrogen) and Alexa Fluor 555 donkey anti-mouse IgG (A31570, Invitrogen), diluted with blocking buffer at 1:1000) at 37 °C for 1 h. Cells were washed again and mounted with Vectashield (H-1800, Vector Laboratories, Burlingame, CA, USA), and the cells were observed under fluorescent microscopy (BZ-X700).