Alexa fluor 594 conjugated goat anti mouse igg

For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde. After washing with PBS, cells were blocked with 0.5% normal goat or donkey serum containing 0.1% Triton X-100, and then treated with primary antibodies. Primary antibodies included anti-Oct3/4 rabbit polyclonal antibody (1∶500; PM048, MBL), anti-Nanog rabbit polyclonal antibody (1∶500; a gift from S. Yamanaka), anti-H3K27me3 rabbit polyclonal antibody (1∶250; 07–449, Millipore), anti-Esrrb mouse monoclonal antibody (1∶500; PP-H6705-00, Perseus), anti-Foxa2 goat polyclonal antibody (1∶1000; sc-9187, Santa Cruz Biotechnology), and anti-Sox17 goat polyclonal antibody (1∶1000, AF1924, R&D Systems). Alexa Fluor 594–conjugated goat anti-mouse IgG (1∶500, Invitrogen), Alexa Fluor 568–conjugated goat anti-rabbit IgG (1∶500, Invitrogen), Alexa Fluor 568–conjugated donkey anti-goat IgG (1∶500, Invitrogen), Alexa Fluor 488–conjugated goat anti-rat IgG (1∶500, Invitrogen), Alexa Fluor 405–conjugated goat anti-mouse IgG (1∶500, Invitrogen), and Alexa Fluor 405–conjugated goat anti-rabbit IgG (1∶500, Invitrogen) were used as secondary antibodies. Nuclei were stained with 1 µg/ml DAPI (Sigma) or 1 µM TO-PRO-3 iodide (Invitrogen). Alkaline phosphatase staining was performed as described previously [32] (link).

TUNEL staining was carried out using a promega apoptosis detection kit. Immunofluorescence for TUNEL staining was performed with Alexa Fluor 594-conjugated goat anti-mouse IgG (1: 500; Invitrogen). The glass was mounted with cover slips containing Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) and imaged under an fluorescent microscope.

NRK52E cells, tissue samples from the patients and rats were labeled with antibodies to CDK5 (1:100), p35 (1:100), ERK1/2 (1:100), pERK1+pERK2 (1:100), PPAR gamma (1:100), pPPAR gamma (1:100), E-cadherin (1:100), Vimentin (1:100) and Collagen IV (1:100). For immunofluorescence staining, Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Cambridge, MA) were used for secondary antibodies, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis., MO) and coverslipped with aqueous mounting medium (CTS011, BD Bioscience, Minneapolis, MN). For immunohistochemistry, EnVision™ Detection Systems Peroxidase/diaminobenzidine (DAB), Rabbit/Mouse kit (K4065, Dako, Carpinteria, CA) was used. Nuclei were counterstained with hematoxylin and coverslipped with Permount mounting medium (00-4960-56, eBioscience, San Diego, CA).
Samples were evaluated semiquantitatively by systematically selecting without bias twenty fields for analysis. Images were taken with a BX51 light microscope (Olympus, Tokyo) with appropriate filters. Staining intensity was measured using Image J analysis software (Image J 1.44, National Institute of Health). PBS instead of primary antibodies served as a negative control.

To verify the identity and assess the purity of isolated glial cells, immunofluorescent microscopy was used. Cells plated on poly-D-lysine-coated coverslips were fixed in acetic acid/ethanol [5:95 (v/v)] at −20 °C for 10 min and then incubated with the relevant primary antibody for 30 min at room temperature, followed by three washes in Minimum Essential Medium (MEM) (Invitrogen) and subsequent incubation with secondary antibody for 30 min. Sections were mounted in a medium containing 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) (100 ng/mL; Vector Laboratories) to show cell nuclei, and examined by confocal microscopy (Olympus FluoView FV10i; Olympus, Tokyo, Japan). The primary antibodies used were anti-GFAP (rabbit polyclonal antibody; 1:1000; Cat#ab7260, Abcam, MA) for astrocytes, anti-GalC (mouse monoclonal antibody; 1:100; Cat#MAB342, Merck Millipore) for oligodendrocytes, and anti-A2B5 (mouse monoclonal antibody; 1:100; Cat#MAB312, Merck Millipore) for OPCs. The secondary antibodies used were Dylight 488-conjugated goat anti-rabbit IgG (1:200; Cat#E032220-2, EarthOx) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200; Cat#A-11005, Invitrogen).

The following antibodies and reagents were used: pα-Syn (Ser129, Biolegend, 825701), pα-Syn (Ser129, Cell Signaling Technology, 23706s), MAP2 (Thermo Fisher Scientific, SF254293), COX IV (Abcam, ab16056), ATG5 (Proteintech, 10181-2-AP), Beclin1 (Proteintech, 11306-1-AP), LC3 (Cell Signaling Technology, 12741), Bcl2 (Cell Signaling Technology, 3498S), Bax (Proteintech, 50599-2-Ig), GAPDH (Proteintech, 60004-1-Ig), TH (Sigma-Aldrich, AB152), Ubiquitin (Santa Cruz Biotechnology, sc-8017), Iba-1 (Wako, 019-19741), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen, A-11005), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11012), DAPI (Biofroxx, EZ3412B205), HRP-conjugated anti-mouse IgG (BIO-RAD, 170-6516), HRP-conjugated anti-rabbit IgG (BIO-RAD, 170-6515), Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721), and Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1).