4 6 diamidine 2 phenylindole dihydrochloride dapi

The morphological examination of in vitro cultured rBMSCs on all the membranes were performed after 1, 4, and 7 days of cell culture using SEM and confocal laser scanning microscopy (CLSM). The samples for SEM were prepared as previously indicated in Section 2.2.2. For CLSM (IX-71, Olympus Co., Ltd., Shinjuku, Tokyo, Japan) exanimation, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min at room temperature, and then stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich Co., LLC., Rockville, MD, USA) for nucleus and Alexa Fluor 546 phalloidin (Invitrogen, CA, USA) for F-actin.

Nardosinone (Figure 1A), purchased from Must Biotechnology (Chengdu, China), was dissolved in dimethyl sulfoxide (DMSO). Alpha modification of Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, United States). Recombinant murine M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, United States). Tartrate-resistant acid phosphatase (TRAP) staining solution, Triton X-100, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). FITC phalloidin was obtained from Yeasen biotech Co., Ltd (Chengdu, China). Primary antibodies targeting GAPDH, IκBα, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, c-Fos and NFATc1 were purchased from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kits were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). LPS from P. gingivalis was purchased from InvivoGen (San Diego, CA, United States).

The NAMs used in
this work consisted of a 6-carboxyfluorescein-labeled 14-mer oligonucleotide
formed by an LNA, 2′OMe, and PS linkage (Eurogentec, Seraing,
Belgium). This sequence was designed to target a conserved region
of the 16S rRNA in the domain Eubacteria.^{52 (link)} The synthetic phospholipids 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP, chloride salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE, chloride salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG, ammonium
salt), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) were purchased
from Avanti Polar Lipids (Alabaster, AL, USA). 4′,6-Diamidine-2′-phenylindole
dihydrochloride (DAPI) was purchased from Sigma-Aldrich (Lisbon, Portugal).
All other reagents were of analytical grade.

Functional status of mitochondria was evaluated by MitoTracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. M7512) dye, depending on mitochondrial membrane potential of living cells. hCmPCs were seeded in Nunc Lab-Tek chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 5.0 × 10⁴/well in 500 μL culture medium, incubated overnight at 37 °C and then for 30 min with the MitoTracker Red CMXRos at final concentrations of 200 nM in serum-free medium. After cell fixing in 4% formaldehyde, the fluorescent dye 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. 10236276001) was applied for 6 min (dilution 1:20). Fluorescence of mitochondrial dye was imaged using a Zeiss LSM 780 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) and quantitative analyses were performed using ImageJ software (NIH), expressed as percent (%) fluorescent intensity (red) [57 (link)].

Immunohistochemistry of SVZ and OB was performed on 40-μm serial free-floating sections. To improve the efficiency of BrdU detection, sections and cells, prior to antibody staining, were exposed to 2 N HCl for 30 min (for sections) or 15 min (for cells), respectively, at 37 °C and then washed with 0.1 M sodium borate buffer pH 8.5 for 10 min (to allow the denaturation of DNA, necessary to expose the BrdU). Upon fixation, sections or cells were permeabilized in blocking solution (0.3 or 0.1% Triton X-100, respectively, in PBS, 3 or 10% normal donkey serum and normal goat serum, respectively) and then incubated with the antibody of interests (Table I). The total number of cells in each field was determined by counterstaining cell nuclei with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich; 50 mg/ml in PBS for 15 min at RT). Immunostained sections and cells were mounted in Aqua-Poly/Mount (Polysciences) and analyzed at epi-fluorescent or confocal microscopy, using a Nikon Eclipse 90i microscope (Nikon) or a TCS SP5 microscope (Leica Microsystem). Z-stacks images were captured at 1 μm intervals with a ×40 or ×63 objectives and a pinhole of 1.0 Airy unit. Analyses were performed in sequential scanning mode to rule out cross-bleeding between channels. Fluorescence intensity quantification was performed with ImageJ software.

Inflammasome complex assembly was evaluated by the detection of “specks" formation using immunofluorescence microscopy. MDM were fixed and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) for 30 min at 37°C and 5% CO₂ and incubated with primary antibody for NLRP3 (1:100 mouse anti-human NLRP3, Abram) and/or NLRC4 (1:200 rabbit anti-human NLRC4; Biolegend) overnight at room temperature. Fluorescent secondary antibodies (Alexa 488-conjugated goat-anti-mouse IgG1, or Alexa 647-conjugated goat-anti-rabbit IgG1; Thermofisher Scientific) were then added for 1 h. 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) was used for nuclear counterstaining. Image acquisition was performed at the microscope facility at the Laboratory of Cellular Biology from the Butantan Institute (São Paulo, SP, Brazil) using a DMi8 confocal laser scanning microscope equipped with a digital camera DFC310 FX (Leica). The counting of NLRP3+ and NLRC4+ specks in MDM was performed manually by observing speck formation within the cells in 10 fields (26 (link)).