Journal: Frontiers in Medicine

Article Title: Immunocytochemical examination of Akt, mTOR, and Pax-2 for endometrial carcinoma through thin-layer endometrial cytology

doi: 10.3389/fmed.2025.1576060

Figure Lengend Snippet: The scatterplot of biomarkers in IHC and ICC. (A) Scatterplot for AKT endometrial expression. (B) Scatterplot for mTOR endometrial expression. (C) Scatterplot for Pax-2 endometrial expression. IHC, immunohistochemistry; ICC, immunocytochemistry.

Article Snippet: The assays were conducted in a humidity-controlled environment as follows: (1) sections were incubated with ethylenediaminetetraacetic acid (EDTA) (pH8.0); (2) sections were rinsed three times with PBS; (3) sections underwent overnight incubation with rabbit anti-human AKT antibody (#9611, Cell Signaling TechnologyTM, USA, 1:600 dilution), rabbit anti-human mTOR-related protein antibody (#2983, Cell Signaling TechnologyTM, USA, 1:150 dilution), and rabbit anti-human Pax-2 antibody (ZSGB-BIOTM, Beijing, China); (4) sections were rinsed four times with PBS (5 min per rinse); (5) ChemMateTM EnVisionTM/HRP (DakoTM, Herndon, VA, USA) was introduced, followed by incubation of the sections for 30 min at 37°C; (6) sections were rinsed four times with PBS; (7) staining was performed using 3,3-diaminobenzidine (DAB) at room temperature in the dark for 180 s; (8) distilled water halted the DAB staining; (9) sections were hematoxylin-stained; and (10) sections were dehydrated, cleared, and mounted with neutral-gum, with positive controls treated in the same manner.

Techniques: Expressing, Immunohistochemistry, Immunocytochemistry

Journal: Frontiers in Medicine

Article Title: Immunocytochemical examination of Akt, mTOR, and Pax-2 for endometrial carcinoma through thin-layer endometrial cytology

doi: 10.3389/fmed.2025.1576060

Figure Lengend Snippet: ROC curve of biomarkers in IHC and ICC. (A) ROC curve for AKT. (B) ROC curve for mTOR. (C) ROC curve for Pax-2. The green line indicates ICC, and the blue line represents IHC. IHC, immunohistochemistry; ICC, immunocytochemistry.

Article Snippet: The assays were conducted in a humidity-controlled environment as follows: (1) sections were incubated with ethylenediaminetetraacetic acid (EDTA) (pH8.0); (2) sections were rinsed three times with PBS; (3) sections underwent overnight incubation with rabbit anti-human AKT antibody (#9611, Cell Signaling TechnologyTM, USA, 1:600 dilution), rabbit anti-human mTOR-related protein antibody (#2983, Cell Signaling TechnologyTM, USA, 1:150 dilution), and rabbit anti-human Pax-2 antibody (ZSGB-BIOTM, Beijing, China); (4) sections were rinsed four times with PBS (5 min per rinse); (5) ChemMateTM EnVisionTM/HRP (DakoTM, Herndon, VA, USA) was introduced, followed by incubation of the sections for 30 min at 37°C; (6) sections were rinsed four times with PBS; (7) staining was performed using 3,3-diaminobenzidine (DAB) at room temperature in the dark for 180 s; (8) distilled water halted the DAB staining; (9) sections were hematoxylin-stained; and (10) sections were dehydrated, cleared, and mounted with neutral-gum, with positive controls treated in the same manner.

Techniques: Immunohistochemistry, Immunocytochemistry

Journal: Autophagy

Article Title: Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2 R1441G mice

doi: 10.1080/15548627.2020.1850008

Figure Lengend Snippet: Accumulation of ubiquitinated mitochondria in aged LRRK2 R1441G mutant mouse striata compared to levels in age-matched wild-type (WT) controls. (a) Representative flow cytometry outputs of isolated mitochondria from 6- and 24-month-old WT and LRRK2 R1441G mutant mouse striatum co-stained with MitoTracker Red (Texas Red channel) and anti-ubiquitin antibody (FITC channel). A total of ~65 µg of freshly isolated mitochondria were obtained from each 6- and 24-month-old WT and LRRK2 mutant mouse whole striatum, and were resuspended in 125 µl suspension buffer for staining. (b) Relative amount of ubiquitinated mitochondria (Q2) was significantly higher in aged mutant mice (N = 3). (c) Representative immunoblot showing levels of ubiquitinated protein in isolated striatal mitochondrial lysates in 24 month-old WT and mutant mice (WT: wild-type; RG: LRRK2 R1441G mutant); COX4 levels indicate loading of mitochondria; Densitometry analysis showing higher level of ubiquitination in striatal mitochondria of aged mutant mice compared with age-matched WT controls. Data represents mean ± standard error of mean (SEM) from three independent experiments (N = 5). Statistical significance between groups was analyzed by unpaired Student’s t-test. * represents statistical significance at p < 0.05. The “N” represents the number of mice used for measurements. (d) Western blot of mitochondrial lysates to demonstrate the purity of mitochondrial isolates. Mitochondrial lysates showed highly enriched mitochondrial marker protein, VDAC, whereas only trace amounts of lysosomal LAMP2A and ACTB protein were detected in the isolates. (e) MitoTracker Red staining efficiency in isolated mitochondria from aged WT and mutant mice. Total mitochondria were stained against a mitochondrial specific marker protein, VDAC (voltage-dependent anion channel), and subsequently stained with MitoTracker Red. The percentage of double-stained mitochondria (Q2) between WT and mutant mice were similar

Article Snippet: Equal amount (65 μg) of freshly isolated mitochondria from 6- and 24-month-old WT and homozygous LRRK2 mutant mouse whole striatum were resuspended in 125 μl mitochondrial suspension buffer, and immuno-stained with rabbit anti-ubiquitin antibody (1:125; Cell Signaling TechnologyTM, 3933 at room temperature for 1.5 h, followed by incubation with F(ab’)2 goat anti-rabbit IgG (H + L) cross-adsorbed Alexa-Fluor® 488 secondary antibody (1:300; ThermoFisher ScientificTM, A-11,017) and 50 nM MitoTrackerTM Red CMXRos (ThermoFisher ScientificTM, M7512) at room temperature for 0.5 h. Both the antibodies were diluted in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4 (ThermoFisher ScientificTM, AM9624). containing 5% normal serum (ThermoFisher ScientificTM, 16,210,072) and 5% BSA (Sigma AldrichTM, A9647).

Techniques: Mutagenesis, Flow Cytometry, Isolation, Staining, Ubiquitin Proteomics, Suspension, Western Blot, Marker