Alexa fluor 568 goat anti rabbit antibody

Tissue samples were fixed in 4% PFA, cryopreserved, and sectioned as previously described [12 (link)]. For immunolabeling, sections were permeabilized with 0.5% Triton-X/PBS for 10 min at room temperature and incubated in 2% BSA/5% normal goat serum/0.1% Triton-X/PBS for 30 min at room temperature to block non-specific sites. Samples were then incubated overnight at 4 °C with primary antibodies diluted in a blocking solution, washed with 1 x PBS, and incubated for 1 h at room temperature in Alexa Fluor 568 Goat anti-Rabbit antibody (Thermo Fisher Scientific) diluted 1:1000 in serum-free blocking solution. Nuclei were counterstained with DAPI (1:1000 in PBS). Images were collected using a Nikon TE2000 confocal microscope. All tissues to be compared were immunostained at the same time, and images were captured the same day under the same laser settings to avoid interexperimental variability. The following antibodies were used in this study: anti-GFAP (AB53554 from Abcam), anti-cCASP3 (Asp175, Cell Signaling).

Heads of adult mice were decalcified with Kalkitox (Wako, Osaka, Japan) overnight at 4 °C and neutralized with 5% sodium sulfate solution (Wako) for more than 2 h. Paraffin-embedded sections were subjected to immunostaining with primary anti-Sox2 rabbit monoclonal antibody (1:100) (ab92494; Abcam, Cambridge, UK), and secondary anti-rabbit antibody (414341; Nichirei Bioscience, Tokyo, Japan), and were counterstained with hematoxylin (Wako). Some sections were immunostained with a primary anti-amelogenin antibody (1:500) (Hokudo, Sapporo, Japan) and a secondary Alexa Fluor 568 goat anti-rabbit antibody (A11036; Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, sections were counterstained with Hoechst 33258 (H3569; Thermo Fisher Scientific). Other sections were immunostained with a primary anti-decorin goat polyclonal antibody (N-15, sc-22613; Santa Cruz Biotechnology, Dallas, TX, USA), and a secondary anti-goat antibody (414351; Nichirei Bioscience); and diaminobenzidine (415171; Nichirei Bioscience). The other sections were immunostained with a primary anti-N-cadherin Rabbit polycloneal antibody (1:100) (ab18203; Abcam) and a EnVision System HRP-labeled polymer (rabbit, K4003; Dako) with diaminobenzidine (K3468; Dako). They were also counterstained with hematoxylin.

HaCaT cells (2 × 10⁴ cells/well) were plated in a 24-well plate and cultured for 24 h. The cells were rinsed with PBS; treated with a medium containing vehicle, IPE, or GR inhibitor (mifepristone; Sigma-Aldrich, St. Louis, MO, USA) for 1 h; and stimulated with cortisol for 1 h. The cells were fixed with 4% formaldehyde solution (Biosesang, Seongnam, Republic of Korea), permeabilized with 0.1% Triton X-100, and blocked with 5% FBS and 1% BSA solution for 1 h. The cells were then sequentially incubated with polyclonal anti-rabbit antibody against the GR (1:20; Abcam, Cambridge, U.K.) and Alexa Fluor™ 568 goat anti-rabbit antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA). The nuclei of HaCaT cells were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA), and immunofluorescence was examined using the EVOS TM FL Auto2 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). GR translocation was analyzed using images merged with GR and DAPI.

SOX2 (D6D9), OCT4 (C30A3), γH2AX (Ser139), CDK1, CDK2, p15INK4B, Cyclin D1 (DSS6), p-Aurora A (T288), p-Aurora B (T232, 1:1000), Aurora B, c-Myc (9E10) and Phospho-Chk2 (Thr68), Phospho-ATM (Ser198), Phospho p53 (Ser 15) antibodies were purchased from Cell Signalling Technology. GFP (clone B-2) antibody was purchased from Santa Cruz Biotechnology. Primary antibodies are used in 1:200 dilution for Immunofluorescence assay and 1:1000 dilution for Western blotting. GAPDH (clone MAB374, 1:4000) antibody was purchased from Millipore.
Horse radish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000) were purchased from Jackson ImmunoResearch. Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 568 goat anti-rabbit antibody (1:200) were purchased from Life Technologies. Anti-PRL3 monoclonal antibody (mAb) (clone 318, 1:200 for Immunofluorescence and 1:2000 for western blot) was generated in-house. PRL3-zumab was engineered based on the original framework of murine anti-PRL3 mAb (clone 318).

Ae. aegypti Liverpool, CRVP379-pKO, and CRVP379-cKO males and females were cold-anesthetized on ice. Ovaries and testes from four-day old mosquitoes were dissected using sharp forceps and entomological needles. The ovaries were stained following a method previously described [45 (link)]. CRVP379 antibody was used at a dilution of 1:200, and AlexaFluor568-goat anti-rabbit antibody (Life Technologies, A11011) was used at a dilution of 1:200. Samples were mounted using Fluoromount-G with DAPI (Invitrogen, 00-4959-52) and examined with a Zeiss LSM 1710 confocal microscope. Images were analyzed with ImageJ Fiji software [46 (link)]. The Z-stack (all slides) data sets were analyzed and presented as a single 2D projection. Brightfield and DAPI stained ovaries were also observed under a LEICA DM2500 microscope, and images were captured with a DFC310 FX camera. Three ovaries from each line were used to measure the follicular cell size using ImageJ Fiji software, License GPLv3+, Release 2.5.0.

Cholesterol, U18666a, sodium taurocholate, mono-olein, oleic acid, phosphatidyl-choline (2-oleoyl-1-palmityl-sn-glycero-3-phosphocholine) were from Sigma-Aldrich (St. Louis, MO); ezetimibe and lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphocholine) were from Santa Cruz Biotechnology (Santa Cruz, CA); Ni-NTA resin was from Qiagen (Valencia, CA), NHS-activated Sepharose 4 Fast Flow and Q-Sepharose Fast Flow was from GE Healthcare Life Sciences. ³H-Cholesterol was from American Radiolabeled Chemicals (St. Louis, MO) and ³H-ezetimibe was from Merck. EZ-link Sulfo-NHS-SS-Biotin, SF900 III SFM insect cell medium, Freestyle 293 expression medium, Dulbecco’s modified Eagle’s medium (DMEM) and neutravidin agarose were from Life Technologies (Carlsbad, CA); lipoprotein-deficient serum was from KALEN Biomedical (Montgomery Village, Maryland). Chicken anti-GFP antibodies were from Life Technologies (used at 1:1000 for immunoblots). IRDye 680RD donkey anti-chicken and IRDye 800CW streptavidin were from LI-COR (Lincoln, NE) and used at 1:10,000 for immunoblots. Mouse anti-GFP antibody was from NeuroMab and Rabbit anti-LAMP1 antibody was obtained from Novus; both were used at 1:1000 for immunofluorescence. Alexa Fluor 488 Goat anti-mouse antibody and Alexa Fluor 568 Goat anti-rabbit antibody were obtained from Life Technologies and used at 1:2000 for immunofluorescence.