Alexa fluor 594 conjugated antibody

PPP6C antibody (rabbit, A300-844A; Bethyl Laboratories, Inc.); SYCP3 antibody (rabbit, NB300-231; Novus Biologicals); α-tubulin antibody (rabbit, 2144; Cell Signaling Technology, Inc.); β-actin antibody (mouse, 3700; Cell Signaling Technology, Inc.); Phospho-β-Catenin (Ser552) (D8E11) antibody (rabbit, 5651; Cell Signaling Technology, Inc.); SYCP3 antibody (mouse, sc-74569; Santa Cruz); γH2AX antibody (rabbit, 9718; Cell Signaling Technology, Inc.); MVH antibody (mouse, ab27591; abcam); SYCP1 antibody (rabbit, ab15090; abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); β-catenin antibody (rabbit, 51067-1-AP, Proteintech); ZO2 antibody (rabbit, 18900-1-AP, Proteintech); TEX14 antibody (rabbit, 18351-1-AP, Proteintech); Vimentin antibody (rabbit, 10366-1-AP, Proteintech); HA-Tag mab (mouse, AE008; ABclonal); c-Myc antibody (mouse, m4439; sigma); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo). Horseradish peroxidase–conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488–conjugated antibody and Alexa Fluor 594–conjugated antibody were purchased from Life Technologies.

Digoxygenin (DIG)-labeled DmCycB probes were synthesized for fluorescence in situ hybridization. According to a previously described procedure (35 (link)), the isolated salivary glands were fixed with 4% paraformaldehyde and then permeabilized in PBST buffer for 30 min. Following the prehybridization process in hybridization buffer (5× SSC and 50% deionized formamide), samples were incubated with DIG-labeled probes for 12 h at 56°C. After a series of washes to significantly decrease background, the salivary glands were incubated with rabbit anti-DIG antibody (1:1000; Invitrogen) for 2 h and then with an anti-rabbit Alexa Fluor 594-conjugated antibody (1:1000; Life Technologies). DAPI (1:1000; Thermo Fisher Scientific) was used for nuclear labeling. The signals were captured by confocal microscope (Olympus Fv1000). The primers used to prepare the DIG probes are listed in Supplementary Table S1.

In order to visualize vimentin, 4 × 10⁴ HBECs were seeded on sterile cover slips. PKH67 green fluorescent labeled exosomes were added to the cells and incubated for 16 hrs. Cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature; immunofluorescence was performed to detect Vimentin. Briefly, cells were blocked and incubated with anti-Vimentin (1:100, Santa Cruz). The secondary antibody utilized was anti-rabbit Alexa Fluor 594–conjugated antibody (1:2000; Invitrogen). DAPI (Invitrogen, Cat. No D21490) was used for nuclear staining. The cover slips were subsequently mounted onto slides with mounting media (Aqua poly mount, Polysciences) and analyzed via confocal fluorescence microscopy (Olympus Fluoview, FV1000 Spectral Confocal). Positive and negative controls were included on all experiments.

The cells, seeded on coverslips, were fixed in 4% buffered paraformaldehyde solution, permeabilised using 0.1% TritonX-100, incubated overnight with rabbit anti-pH3 antibody 04-817 (Millipore), washed with PBS containing 5 mM EGTA and incubated with goat anti-rabbit Alexa Fluor 594-conjugated antibody A11012 (Invitrogen) for 1 h at RT. For subsequent staining of tubulin, cells were washed with PBS-EGTA, incubated with mouse anti-tubulin antibody T5168 (Sigma) for 1 h at RT, washed with PBS-EGTA and incubated with goat anti-mouse Alexa Fluor 488-conjugated antibody A11029 (Invitrogen). For counterstaining of cell nuclei, the coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Cells were examined using an Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany).

In situ immunofluorescence analysis was performed on H226 cells transduced with SLITRK3 or GFP that were plated on glass cover slips and cultured with or without NTF3. Cells were washed three times with PBS, fixed for 15 min in 5% formaldehyde in PBS solution, rehydrated by three washes with PBS, pre-incubated 10 min in 2% BSA-PBS solution, and incubated 45 min at room temperature with primary antibody against NTRK3 (product no. 3376S; Cell Signaling. Danvers, MA) or phosphorylated NTRK3 (Novus Biologicals, Littleton, CO; product no. NBP1–03448) diluted 1:200 in 2% BSA-PBS solution. Bound antibody was detected by staining with a secondary goat anti-rabbit Alexa Fluor 594-conjugated antibody (Invitrogen, Carlsbad, CA) diluted 1:500 in 2% BSA-PBS solution, and incubated for 45 min at room temperature, followed by 3 times of washing with 0.05% Tween-20/PBS solution. Cover slips were fixed to slides with a drop of ProLong Gold Anti-fade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei (Invitrogen). Images were recorded at 40× magnification using an EVOS Digital Inverted Microscope (Advanced Microscopy Group, Mill Creek, WA).