Rabbit anti ki67 antibody

In order to obtain accurate data, immunohistochemistry was carefully conducted under the same conditions. Five tissue sections were selected at 180 μm apart between 1.46 and 2.46 mm posterior to the bregma, according to a mouse atlas [22 ]. The sections were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in 0.1 M PBS and 10% normal horse serum in 0.1 M PBS. Subsequently, the sections were incubated with diluted rabbit anti-Ki67 antibodies (1:500; Abcam, Cambridge, UK) or goat anti-doublecortin (DCX) antibodies (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight, and they were then exposed to biotinylated goat anti-rabbit or rabbit anti-goat IgG (1:400; Vector Labs., Burlingame, CA, USA) and streptavidin-peroxidase complex (1:400; Vector Labs., Burlingame, CA, USA). The sections were visualized by a reaction with 3,3’-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA).

Vps35 mutant mice have been described previously [13 (link), 21 (link), 25 (link)]. Mice were maintained on a standard rodent diet and in a standard facility at Augusta University. Animal care was approved by the Institute of Animal Care and Use Committee (IACUC) at the Augusta University according to the National Institute of Health guidelines.
The following reagents were used: mouse anti-Neuronal Class III ß-Tubulin (Thermo Fisher Scientific, Catalog #:32–2600); rabbit anti-Ki67 antibodies (Abcam, Catalog #: ab15580); chicken anti- ß-galactosidase antibody antibodies (Abcam, Catalog #: ab9361) rabbit anti-SLC4A11 antibodies (Thermo Fisher Scientific, Catalog #: PA5-53730). Rabbit polyclonal anti-Vps35 antibody was generated against the murine Vps35 C-terminal sequence by Cocalico Biologicals, Inc. (PA, USA) as described previously [13 (link)]. Phalloidin-fluorescent conjugates were purchased from Molecular Probes (Thermo Fisher Scientific, Catalog #:A12379). For immunofluorescence analysis, the secondary antibodies were Alexa Fluor-488 or Alexa Fluro-594 conjugated anti-mouse or anti-rabbit antibodies (Invitrogen). For Western analysis, the secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Santa Cruz Biotechnology, Inc.). All cell culture reagents were purchased from Thermo fisher.

All sections were processed under the same conditions to ensure that the immunohistochemical data were comparable among the groups. Tissue sections located at a distance of 90 μm from each other were selected from an area between 1.82 and 2.30 mm posterior to the bregma, as defined by a mouse atlas [41 ] for Ki67, DCX, and pCREB immunohistochemistry. The sections were sequentially treated with 0.3% H₂O₂ in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min at 25 °C. Sections first underwent an overnight incubation with mouse anti-HSP70 antibody (1:500; Calbiochem, EMD Millipore, Temecula, CA, USA), rabbit anti-Ki67 antibody (1:1000; Abcam), rabbit anti-DCX (1:5000; Abcam), or rabbit anti-pCREB (1:400; Cell Signaling Technology, Inc.) at 25 °C. Thereafter, the sections were treated with biotinylated goat anti-mouse or anti-rabbit IgG and a streptavidin-peroxidase complex (1:200; Vector, Burlingame, CA, USA) for 2 h at 25 °C. Sections were visualized by reaction with 3,3′-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. Sections were dehydrated and mounted with Canada balsam (Kanto Chemical, Tokyo, Japan).

Apoptotic changes and cell proliferation were measured using flow cytometry, as described previously [23 (link)]. Briefly, for apoptosis, cell samples were incubated with a rabbit anti-annexin V–FITC (1:1000, #14085, Abcam, Cambridge, UK) for 30 min, followed by propidium iodide at 50 μg/mL (Life Technologies, Carlsbad, CA, USA) for 10 min. For determining cell proliferation, cells were fixed in 4% formaldehyde solution, and cell membranes were permeated with saponin at 1 mg/mL (TCI chemicals). The cells were incubated with a rabbit anti-Ki-67 antibody (1:100, #15580, Abcam), and then with an Alexa 488-conjugated goat anti-rabbit IgG antibody (1:1000, #11008, Life Technologies) for 30 min each. All steps were performed on ice, and cells were washed three times with phosphate-buffered saline containing 2% FBS between each step. The cells omitting the primary antibodies were used as negative controls. The immunopositive cells were analyzed on a BD Accuri C6-Plus flow cytometer (BD Bioscience, San Jose, CA, USA).

Cells were collected using Accutase (STEMCELL Technologies Inc., Vancouver, Canada), and their apoptosis and proliferation were measured. Apoptotic cells were measured using annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Briefly, the prepared cells were stained with an anti-annexin-V-FITC antibody (Abcam, Cambridge, UK, dilution of 1 : 1000) for 30 min and then with PI (Life Technologies, Carlsbad, CA, USA) at 50 μg/ml for 10 min. For proliferative cell measures, the cells were fixed in cold methanol for 10 min, and the cell membranes were permeated by treatment with saponin (TCI Chemicals) at 1 mg/ml for 30 min. The cells were incubated with a rabbit anti-Ki67 antibody (Abcam, dilution of 1 : 100) for 30 min and then with an Alexa 488-conjugated goat anti-rabbit IgG antibody (Life Technologies, dilution of 1 : 1000). All steps were performed on ice, and the cells were washed five times with phosphate-buffered saline (PBS) containing 2% FBS between each step. Cell fluorescence was analyzed using a BD Accuri C6 Plus flow cytometer (BD Bioscience, San Jose, CA, USA).