methyl green solution  (Beyotime)

Journal: Current Research in Microbial Sciences

Article Title: Protrichocysts: a hybrid defense extrusive organelle bridging mechanical projection and chemical secretion in ciliates

doi: 10.1016/j.crmicr.2025.100539

Figure Lengend Snippet: Scanning electron micrographs of protrichocysts of Pseudourostyla cristata in the resting state ( A ) and during ejection ( B‒I ) after induction by a methyl green pyronin treatment (see main text for details). (A) Resting state protrichocysts beneath the pellicle when cell was pricked after SEM preparation; a clear cap structure can be observed (black arrow). (B) An extruded protrichocyst with the tip (T) exposed out of the pellicle, surrounded by the cap . (C, D) Protrichocysts with the body partially ejected, showing a loose microtubule-like structure of the cap (black arrows). (E, F) The less dense posterior part of the body exploding on the cell surface (black arrow) and the microtubule-like circles of the cap gradually decomposing, before protrichocysts leave the cell (black arrow). (G, H) After the explosion of the less dense posterior part of the body (black arrow), the shaft is pushed out from it and wrapped around by the dense anterior part of the body (white arrow). (I) The dense anterior part of the body disintegrates, and the tip (arrowhead) is about to disconnect: a filamentous connecting structure is visible (white arrow); (J) The tip (arrowhead) separates from the shaft. Scale Bars = 5 μm ( F ); 3 μm ( A, D, E, G ); 1 μm ( H, I ); 0.5 μm ( B, C ); 0.1 μm ( J ).

Article Snippet: These were: (1) a solution of 11 mg/mL calcium chloride and 0.0025 % alcian blue 8GS (Solarbio, China); calcium chloride can induce the discharging and the alcian blue concurrently stains protrichocysts allowing to perform an additional histochemical analysis ( ; ); (2) a 30 mg/mL methyl green-pyronin solution (Sangon Biotech, China) , which induces the most violent ejection (i.e., with the maximal ejection distance and a full morphological extension of the extrusome) and therefore allows further SEM observation of morphological details during the ejecting process of protrichocysts; (3) a 0.1 mg/mL acridine orange (AO) solution (Sangon Biotech, China) , which keeps the best integrity of the ejected contents and therefore was used for further protrichocyst protein acquisition and HPLC-MS/MS (see below); AO concurrently stains the protrichocysts, allowing to perform an additional histochemical analysis.

Techniques:

Journal: Current Research in Microbial Sciences

Article Title: Protrichocysts: a hybrid defense extrusive organelle bridging mechanical projection and chemical secretion in ciliates

doi: 10.1016/j.crmicr.2025.100539

Figure Lengend Snippet: Bright field (A-G, J) and fluorescence microscope (H, I, K, L) micrographs of protrichocysts extrusion and components in Pseudourostyla cristata after treatment with different chemicals. Ejection induction by methyl green-pyronin ( A, B, G-I ), calcium chloride-alcian blue ( C, D-F) , and acridine orange (AO) ( J-L ) respectively (see main text for details on the treatments). ( A ) methyl green pyronin induced a more violent protrichocysts ejection, namely with the maximal ejection distance and a full morphological extension of the extrusome; black arrow indicates violently ejected protrichocysts. (B, C) Ejected protrichocysts are away from the cell with an exploded body (arrowheads); white arrows indicate the central shaft maintaining the same length as in resting state, while black arrows indicate the spherical anterior ends depolymerized. (D‒F) Protrichocysts’ ejection stimulated by calcium chloride-alcian blue, to show that the spherical anterior end (black arrows) was stained while the body (white arrows) was not. (G‒I) After methyl green-pyronin treatment, FLUTAX-II labeling showing the presence of an acidic component and microtubules in the anterior parts of protrichocysts (black arrows) ejected, but not in the body part (white arrows). ( J ) Aggregated caps of protrichocysts after AO-induced ejection are indicated by black arrows. (K, L) AO treatment labeled the entire protrichocyst, including the cap (black arrows), the body (white arrows), and the central shaft (arrowheads), indicating the presence of an acidic and/or a phosphorylated protein component. Channels: BF, bright field; WG, ex/em: 492/520 nm; WU, ex/em: 460/650 nm. Scale Bars = 50 μm ( A ); 10 μm ( B‒D, G‒L ); 5 μm ( E, F ).

Article Snippet: These were: (1) a solution of 11 mg/mL calcium chloride and 0.0025 % alcian blue 8GS (Solarbio, China); calcium chloride can induce the discharging and the alcian blue concurrently stains protrichocysts allowing to perform an additional histochemical analysis ( ; ); (2) a 30 mg/mL methyl green-pyronin solution (Sangon Biotech, China) , which induces the most violent ejection (i.e., with the maximal ejection distance and a full morphological extension of the extrusome) and therefore allows further SEM observation of morphological details during the ejecting process of protrichocysts; (3) a 0.1 mg/mL acridine orange (AO) solution (Sangon Biotech, China) , which keeps the best integrity of the ejected contents and therefore was used for further protrichocyst protein acquisition and HPLC-MS/MS (see below); AO concurrently stains the protrichocysts, allowing to perform an additional histochemical analysis.

Techniques: Fluorescence, Microscopy, Staining, Labeling

Journal: Frontiers in Medicine

Article Title: Quercetin alleviates rifampicin-induced hepatocyte injury by modulating the Hippo-YAP signaling pathway

doi: 10.3389/fmed.2025.1707248

Figure Lengend Snippet: Quercetin improved mitochondrial membrane potential (MMP) and inhibited Rifampicin (RFP)-Induced apoptosis in HepaRG Cells. (A) CCK-8 experiment to determine the optimal Quercetin concentration for enhancing HepaRG cell viability ( n = 6). (B) CCK-8 assay to assess the protective effect of Quercetin (15 μm) on cell viability in RFP (25 μm)-treated HepaRG cells. Verteporfin (2 μm) was used to inhibit YAP ( n = 6). (C) Flow cytometry scatter plots and quantification of apoptosis rates of Annexin V/PI staining ( n = 3). (D) The mitochondrial membrane potential was assessed using tetramethylrhodamine ethyl ester (TMRE) and quantification of relative fluorescence intensity ( n = 3). Scale bars: 100 μm. Data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Tukey's post-hoc test was applied for multiple group comparisons for Type I error.

Article Snippet: Mitochondrial membrane potential (MMP) was evaluated using tetramethylrhodamine ethyl ester (TMRE) staining (C2001S, Beyotime, China).

Techniques: Membrane, CCK-8 Assay, Concentration Assay, Flow Cytometry, Staining, Fluorescence