Rabbit anti cdk5

For Western blot analyses, the following primary antibodies were used: mouse anti-PSD-95 (1:250; BD transduction), rabbit anti-CDK5 (1:500; Santa Cruz biotechnology, Dallas TX), mouse anti-Akt (1:1000, Cell signal technology, Danvers, MA), rabbit anti-p44/42 MAPK (Erk1/2) (1:1000; Cell signal technology), mouse anti-GFAP (1:500, Sigma Aldrich, Oakville, Ontario), mouse anti-syntaxin (1:10 000, Sigma Aldrich), rabbit anti-GluR1 (1:2000, Millipore, Billrica, MA), mouse anti-GluR2 (1:2000, Millipore), mouse anti-NR1 (1:2000), rabbit anti-D2R (1:500, Millipore) and mouse anti-actin (1:10 000; Millipore). Secondary antibodies IRDye 680 Goat Anti-Rabbit IgG (1:10 000; Mandel Scientific, Guelf, Ontario) or IRDye 800 Goat Anti-Mouse (1:10 000; Mandel Scientific) were then used.
For immunochemistry analysis, mouse monoclonal anti-neuronal nuclei (NeuN) (1:250; Millipore) and mouse monoclonal anti-actin (1:5000; Millipore) were used as primary antibodies. Revelation of labeling using the Odyssey imager was performed using IRDye 800 Goat Anti-mouse IgG (1:1000) and IRDye 680 Goat Anti-mouse IgG (1:1000) secondary antibodies.

For detection of protein-protein interactions, in situ PLA was performed. The components used (Duolink PLA Technology, Sigma-Aldrich) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe, anti-goat PLA minus probe and Detection Reagents Orange. HUVEC were grown on chamber slides (Ibidi^® μ-Chamber 12 well on glass slides, Martinsried, Germany) till they reached 80% confluence. Following fixation and blocking, the cells were incubated with the primary antibodies: mouse anti-RPTPβ/ζ (1:250, #610180, BD Biosciences), rabbit anti-CDK5 (1:100 in TBS-T; #sc-173, Santa Cruz Biotechnology), mouse anti-CDK5 (1:100 in TBS-T; #H00001020-M01A, Abnova, Taipei, Taiwan), goat anti-p35 (1:100 in TBS-T; #sc-31102, Santa Cruz Biotechnology). Subsequently, the cells were incubated with secondary antibodies conjugated with oligonucleotides, after hybridization and ligation of which, the DNA was amplified resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized with Leica SP5 confocal microscope. Estimation of nuclei and cytoplasm size was performed using the Duolink ImageTool software. To calculate the total number of spots per cell, an algorithmic procedure was used as previously described^{55 (link)}.

To confirm whether Cdk5 immunoreactivity is translocated to nuclei or not following TCI, the sections were assessed via DAPI staining at 1 day after the ischemic surgery. Cdk5 immunofluorescence staining was performed using rabbit anti-Cdk5 (diluted 1:100, Santa Cruz Biotechnology Inc.). The sections were incubated in the primary antibody overnight at room temperature. After washing 3 times for 10 min with PBS, these sections were then incubated in Cy3-conjugated horse anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories Inc.) for 2 h at room temperature. They were then stained with DAPI (10 μM) for 10 min at room temperature in the dark, and then rinsed thoroughly in PBS. The immunoreactions were observed under the confocal MS (LSM510 META NLO, Carl Zeiss, Göttingen, Germany).