Detailed Immunofluorescence Staining Protocol

Immunofluorescence staining on 35-mm glass-bottomed dishs was described previously [11 (link)]. The primary antibodies were used at dilutions recommended by the manufacturers. The antibodies (anti human in all cases) used were mouse anti-Tuj1 (Covance, 1:800), rabbit anti-TH (Pel-Freez, 1:1 000), rat anti-DAT (Chemicon, 1:5 000), rabbit anti-DDC (Chemicon, 1:500), rat anti-Serotonin (Chemicon, 1:200), goat anti-ChAT (Chemicon, 1:100), mouse anti-HN (Millipore, 1:200). The immunostaining was developed with appropriate fluorescent-tagged secondary antibodies (Invitrogen, Carlsbad, CA, USA). In all cases, cellular nuclei were stained with DAPI in VectorShield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were collected with a Ziess or Leica SP5 confocal laser-scanning microscope.

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XinJian L., Qian H., Fang L, & Chuan-Yuan L. (2014). Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression. Science China. Life sciences, 57(9), 867-875.

Publication 2014

mouse anti-Tuj1 rat anti-DAT goat anti-ChAT fluorescent-tagged secondary antibodies VectorShield mounting medium

Antibodies Cellular nuclei Confocal laser scanning microscope Dapi Dilutions Fluorescent antibodies Goat Human Mouse Rabbit Serotonin Vector

Corresponding Organization :

Other organizations : Duke University, Shanghai Jiao Tong University, Duke Medical Center

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Variable analysis

independent variables

Primary antibody dilutions

dependent variables

Immunofluorescence staining pattern

control variables

35-mm glass-bottomed dishes
Cellular nuclei stained with DAPI
Mounting medium (VectorShield)
Imaging with Zeiss or Leica SP5 confocal laser-scanning microscope

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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