4 6 diamidino 2 phenylindole (dapi)

Similar to immunohistochemistry, the location and expression levels of LC3 in the corpus cavernosum were measured by incubating tissue slides with anti-LC3 (1:200, ab48394, Abcam) followed by corresponding FITC-conjugated secondary antibody. In addition, DAPI (4’,6-diamidino-2-phenylindole, Sigma-Aldrich) was used to stain the cell nuclei. Also, the persons performing fluorescence staining had been blinded with regard to group allocation.
ROS in the corpus cavernosum was detected using a fluorescent probe (Dihydroethidium, DHE; Beyotime Biotechnology). The tissue slides were incubated with DHE at room temperature for half an hour. DAPI was used to stain the cell nuclei. Then they were observed under a fluorescence microscope image. The results were analyzed by averaging fluorescence intensity of photographs using Image-Pro Plus software 6.0.
In vitro study, CCSMCs were cultured in 6-well plates. After reaching 70%–80% confluent, they were fixed with 4% formaldehyde and treated with DHE or LC3 immunofluorescence staining in similar protocols of the corpus cavernosum.

Goat serum (10%) was used to block the binding of nonspecific proteins to the sections. Primary antibodies were diluted and incubated with the sections overnight at 4 °C: anti-Aqp1 (Santa Cruz Biotechnology), anti-S100a9 (Proteintech, Rosemont, USA), and anti-Car4 (R&D Systems, Minneapolis, USA). The slides were then washed 3 times with PBS and incubated with secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, USA) for 1 h. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). Images were captured with an IX83 fully automatic inverted fluorescence microscope (Olympus, Tokyo, Japan). Adhesion of neutrophils (S100a9) to Aqp1⁺/Car4^- ECs or Aqp1⁺/Car4⁺ ECs was evaluated by experienced pathologists. For quantification, the numbers of adherent Car4-low ECs and neutrophils per blood vessel were counted. Doublets and triplets were split by considering signal over the average size of a cell. Data were depicted as percentages of Car4-low ECs (number of Car4-low ECs interacting with neutrophils/total number of Car4-low ECs in the blood vessel) and neutrophils (number of neutrophils adhering to Car4-low ECs/total number of neutrophils in the blood vessel) per field. The results represent those from three sections, with nine pictures scanned for each section.

Chondrocytes were treated with interleukin-1β (IL-1β, 200-01 B; Peprotech, London, UK), TRPV4 antagonist GSK205 (616,522; Merck Millipore, London, UK) and agonist GSK1016790 A (GSK101, G0798; Sigma Aldrich, Poole, UK). Antibodies for immunocytochemistry: acetylated α-tubulin (1:2,000, T7451, Sigma Aldrich, Poole, UK) and TRPV4 (1:200, SAB2104243, Sigma Aldrich). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (4′,6-diamidino-2-phenylindole (DAPI), Sigma Aldrich). Antibodies for western blotting: acetylated α-tubulin (1:1,000, T7451, Sigma Aldrich) and α-tubulin (1:1,000, ab4074, Abcam, Cambridge, UK).

Cells were treated with Cy5-CCN2(IV) and examined by a Leica DM-IRB confocal microscope (Leica Microsystems; Wetzlar, Germany) equipped with a X40 oil immersion objective. Nuclei were stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) to control for equal cell density. The absence of the primary antibody was used as a negative control. Fluorophore Cy-5 emitted fluorescence was monitored with a 550 ± 20 nm band pass or a 670 nm long-pass filter, and DAPI was excited using a DIODE laser.

3-month-old adult Tg(mbp:nfsB-egfp)^nk002 were anesthetized with 0.1% MS-222 (Sigma). Some of the trunk were dissected and fixed in 4% paraformaldehyde, cryoprotected in 20% sucrose in 0.1 M PBS (pH 7.4), frozen in Optimal Cutting Temperature Compound (Sakura Finetek, Torrance, CA, USA). Transverse cryosectioning at 10 µm were performed with a cryostat (Leica CM1850, Wetzlar, Germany) and mounted on glass slides. Immunohistochemistry was performed using standard procedures (Huang et al., 2012 (link)). In brief, after drying, sections were incubated with 20% normal sheep serum (NSS) in PBS containing 0.5% Triton X-100 (PBST), followed by overnight incubation at 4°C in an anti-GFP antibody (ab6556, diluted at 1∶1000, Abcam, Cambridge, MA). In the next day, sections were washed extensively with PBST, incubated in an FITC conjugated secondary antibody (diluted at 1∶500, Millipore, Billerica, MA) for 1.5 hrs at room temperature. DAPI (4′,6-diamidino-2-phenylindole, Sigma) was used as a counterstain to label the nuclei. After washing in PBST, sections were sealed with mounting media and glass coverslips.

Whole-mount immunostaining was performed as previously described (Choudhury et al., 2016 (link)). In brief, the internal organs of third instar larvae were dissected in 1X PBS (13 mM NaCl, 0.7 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.4) and fixed in 4% formaldehyde for 20 min at RT. Tissues were washed in 1XPBS followed by 1% Triton X-100 treatment for 20 min. Tissues were washed and incubated in blocking solution (10% Fetal Bovine Serum (FBS), 0.05% Sodium Azide in 1X PBS) for 2 hr at RT and then incubated in primary antibodies at 4°C overnight. Tissues were washed and further incubated with appropriate fluorescent-tagged secondary antibodies for 2 hr, typically. After washing, tissues were incubated in DAPI (4–6-diamidino-2-phenylindole, Sigma-Aldrich, 1 μg/mL) for 10 min and mounted in PromoFluor Antifade Reagent (PK-PF-AFR1, PromoKine) mounting medium and examined using a Leica TCS SP2-AOBS confocal microscope.