Protein block plus serum free

Immunofluorescence double-staining with the combination of α-synuclein and phospho-TDP-43 was performed to confirm colocalization of the two proteins. The deparaffinized and rehydrated sections of the thalamic fasciculus of MSA-13 were pretreated with 95% formic acid for 30 minutes and then steamed in distilled water for 30 minutes. Next, sections were blocked with Protein Block plus Serum Free (DAKO) for 1 hour and incubated with both anti-phospho-TDP43 antibody (1:500) and anti-NACP (1:2000) primary antibodies diluted in with Antibody Diluent with Background-Reducing Components (DAKO) overnight at 4°C. Sections were washed three times with 1×PBS at room temperature, and then incubated with secondary antibodies Alexa Fluor 568 (1:500, Thermo Fisher Scientific, Inc.) and Alexa Fluor 488 (1:500, Thermo Fisher Scientific, Inc.) diluted with Antibody Diluent with Background-Reducing Components (DAKO) for 1.5 hours at room temperature in a dark chamber. Sections were washed three times with 1×PBS at room temperature, incubated with 1% Sudan Black for 2 minutes, washed with distilled water and mounted with Vectashield mounting media containing DAPI (Vector Laboratories). Representative images were taken with a confocal laser-scanning fluorescent microscope (LSM 800; Carl Zeiss, Jena, Germany).