Hrp conjugated anti rabbit and anti mouse secondary antibodies

A complete list of all primary antibodies used in this study can be found in Table S1. Secondary HRP-conjugated anti-mouse and anti-rabbit antibodies for immunoblotting were from GE-Healthcare (Chicago, IL, USA). Secondary AlexaFluor-488, -594 and -647-conjugated anti-mouse and anti-rabbit antibodies and Cy3-conjugated anti-rat antibody for immunofluorescence microscopy, flow cytometry and DNA fiber analysis were from Invitrogen and Jackson ImmunoResearch (West Grove, PA, USA), respectively.

A complete list of all primary antibodies (with manufacturers, catalogue numbers and applications) used throughout this study can be found in Supplementary Table 1. Secondary HRP-conjugated anti-mouse and anti-rabbit antibodies were from GE- Healthcare and the HRP-conjugated anti-goat antibody was from Santa Cruz Biotech. Alexa Fluor-488, -594, and -647-conjugated secondary antibodies were purchased from Invitrogen. Rabbit polyclonal antibodies specific for KLHL15 were generated as follows. Human KLHL15 cDNA corresponding to amino acids 300–604 was cloned into pET30a (Novagen) vector for expression in Escherichia coli. The recombinant His-tagged KLHL15 fragment was purified using Ni-NTA (Qiagen) following the manufacturer's instructions and subsequently used to immunize rabbits. After five immunizations, serum was obtained and purified against the recombinant antigen. For that, 100–200 μg of the KLHL15 antigen was loaded onto SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a nitrocellulose membrane before staining with Ponceau S. The part of the membrane containing the antigen was cut out, blocked with 2% BSA in TBS-T for 1 h and then incubated with the serum overnight at 4 °C. Bound antibodies were eluted with 0.15 M glycine-HCl, pH 2.3. 1 M Tris-HCl, pH 8.8, was immediately added to neutralize the pH of the antibody solution to pH 7.5.

Rabbit anti-MANF antibody was purchased from Abcam (Cambridge, MA). Recombinant MANF protein was expressed and purified as previously described [10] (link). Mouse anti-neuronal nuclei (NeuN) antibody was obtained from Millipore Corporate (Billerica, MA). Mouse anti-glial fibrillary acidic protein (GFAP) and calbindin antibodies were obtained from Sigma Chemical Co. (St. Louis, MO). Mouse anti-tyrosine hydroxylase antibody was purchased from BD biotechnology (San Diego, CA). Biotin-conjugated anti-mouse and anti-rabbit secondary antibodies and the ABC kit were obtained from Vector (Burlingame, CA). Alexa-488 conjugated anti-rabbit and Alexa-594 conjugated anti-mouse antibodies were obtained from Life Technologies (Grand Island, NY). Mouse anti-actin, HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from GE Healthcare Life Sciences (Piscataway, NJ). Ketamine/xylazine was obtained from Butler Schein Animal Health (Dublin, OH). Other chemicals and reagents were purchased either from Sigma Chemical or Life Technologies.