Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: PLX3397 attenuates astrocytic scar formation and increases the inflammatory response following SCI. (A) Representative images of astrocytic scars in sagittal sections of the spinal cord at 14 days after spinal cord injury (SCI). In SCI mice, astrocytes surrounding the lesion area packed tightly together as an astrocytic scar barrier, and this astrocytic front was less prominent in mice with PLX3397 treatment. Scale bars: 50 μm. (B) In the SCI mice, the spread of CD68 + inflammatory cells was restricted by a dense glial scar, whereas the boundary of the PLX3397-treated mice was disrupted, and a significant infiltration of CD68 + cells was observed. Scale bars: 50 μm. (C) aCasp3/NeuN costaining in sagittal sections indicating apoptotic neurons. Scale bars: 50 μm. (D) PLX3397 treatment increased the lesion size (calculated as GFAP-negative staining area). Microglia elimination increased the concentration of the proinflammatory cytokines (E) TNF-α, (F) IL-6, and (G) IL-1β at 14 days post-injury (dpi). (H) The proportion of NeuN + /aCasp3 + cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test for E–G; unpaired t -test for D and H). Data are expressed as the mean ± SD. The experiments were repeated six times. aCasp3: Activated caspase-3; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; IL: interleukin; NeuN: neuronal nuclei; PLX3397: colony-stimulating factor 1 receptor inhibitor; SCI: spinal cord injury; TNF-α: tumor necrosis factor-α.

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: Negative Staining, Concentration Assay

Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Western Blot, Staining

Journal: Gut Microbes

Article Title: Epithelial talin-1 protects mice from citrobacter rodentium -induced colitis by restricting bacterial crypt intrusion and enhancing t cell immunity

doi: 10.1080/19490976.2023.2192623

Figure Lengend Snippet: Loss of epithelial-specific talin-1 suppresses pathogen-induced epithelial apoptosis. (a) Representative images of colon tissues immunoperoxidase-stained for cleaved caspase-3. (b) the proportion of cleaved caspase-3-positive mucosa determined by measuring the total height of the mucosa and the height of the region with positive staining. Each dot represents measurements from a high-powered field; n = 12–15 fields from 3 different mice per group. All values reported with the median depicted as a thick line and the upper and lower quartiles as thin lines. (c) Expression of the gene encoding TNF-α analyzed by RT-qPCR. n = 4 uninfected mice and n = 6–7 infected mice per genotype. Each symbol is a different mouse. Values are reported as mean ± SEM. * P < 0.05 and ** P < 0.01 determined by (b) 1-way ANOVA and Tukey test and (c) 1-way ANOVA with Kruskal-Wallis test, followed by a Mann-Whitney U test. Scale bars represent 100 μm.

Article Snippet: Tissues were then incubated overnight at 4°C using the following antibodies: prediluted rabbit polyclonal anti-Ki-67 (Biocare, PRM325AA), prediluted rabbit monoclonal anti-MPO (Biocare, PP023AA), rabbit polyclonal anti-CD3 (Abcam, ab5690; 1:150), or rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling, 9664; 1:400).

Techniques: Staining, Expressing, Quantitative RT-PCR, Infection, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Mesenchyme-derived vertebrate lonesome kinase controls lung organogenesis by altering the matrisome

doi: 10.1007/s00018-023-04735-6

Figure Lengend Snippet: Loss of mesenchyme-derived VLK causes severe lung abnormalities. A Ratio of lung weight of E18.5 embryos relative to whole body weight. N = 4–5. Each image depicts a single mouse. B , C Representative photomicrographs ( B ; Scale bars: 50 mm) and quantification of positively stained area relative to total lung area in lung sections from E18.5 CTRL and Pkdcc −/− mice using the indicated histological stainings or measuring young collagen based on Herovici stainings ( B ). N = 6 mice per genotype. D–F Representative immunofluorescence stainings of mouse lung sections from E18.5 CTRL and Pkdcc − /− mice for Ki67 ( D , Scale bars: 50 μm) and quantification of Ki67-positive cells per area ( E ) or total nuclei count per field of view ( F ). N = 4–10 mice per genotype. G Representative immunofluorescence stainings of mouse lung sections from E18.5 CTRL and Pkdcc − /− mice for cleaved caspase 3. Liver sections from mice treated with the hepatotoxin CCl 4 were used as a positive control (Scale bars: 100 μm). H RT-qPCR analysis of RNA samples from E18.5 lung tissue of CTRL and Pkdcc −/− mice for Pkdcc , Col3a1 , Col1a1 and Timp1 relative to Rps29 . N = 8 per genotype. I, J Biochemical analysis of matrix proteins ( I ) or collagen cross-links ( J ) in E18.5 lungs. N = 8 per genotype, for each data point two embryos were pooled. Bar graphs indicate mean ± S.D. P values are indicated in the graphs; statistical analysis was performed using Mann–Whitney U test. Each image depicts a single mouse

Article Snippet: The following antibodies were used for immunostaining: rabbit anti-VLK 404 (Whitman laboratory, Harvard University, Boston, MA), goat anti-cytokeratin 19 (Hybridoma Product TROMA-III; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA), rabbit anti-SPC (#sc-13979, Santa Cruz, Santa Cruz, CA), rat anti-Ki-67-FITC (#11-5698-82, Thermo Fisher Scientific), rabbit anti-fibromodulin (#ab81443, Abcam, Cambridge, UK), mouse-anti podoplanin (#8.1.1.; DSHB), rabbit-anti-SOX9 (#AB5535 Millipore, Darmstadt, Germany), rat anti-CD31-phycoerythrin (PE) (#553370, BD Pharmingen, San Diego, CA), rabbit anti-matrilin-4 (#ab106379, Abcam), rabbit anti-cleaved caspase 3 (#9661, Cell Signaling, Danvers, MA), guinea pig anti-pan keratin (GP14, Progen Biotechnik GmbH, Heidelberg, Germany), goat anti-PDGFR alpha (AF1062, R&D Systems, Minneapolis, MN), donkey anti-rabbit-Cy3 (#711-165-152, Jackson ImmunoResearch, West Grove, PA), donkey anti-rat-Cy3 (#712-165-150, Jackson ImmunoResearch), bovine anti-goat-Cy3 (#805-165-180, Jackson ImmunoResearch), goat anti-guinea pig-Cy2 (#106-225-003, Jackson ImmunoResearch) and donkey anti-goat-Cy2 (#705-225-147, Jackson ImmunoResearch).

Techniques: Derivative Assay, Staining, Immunofluorescence, Positive Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancers

Article Title: Evaluation of Electrochemotherapy with Bleomycin in the Treatment of Colorectal Hepatic Metastases in a Rat Model

doi: 10.3390/cancers15051598

Figure Lengend Snippet: ( A – D ) Immunohistochemical analysis of cleaved caspase-3 in animals treated with ECT ( A ), rEP ( B ), BLM ( C ), or Sham ( D ). Apoptotic cells (arrows) are stained red. Scale bars: 50 µm. ( E ) The diagram displays the mean number of positive cells in the tumor tissue per HPF. Data are given as mean ± SEM; § p < 0.05 vs. Sham.

Article Snippet: For the immunohistochemical detection of apoptotic cells, sections were stained with a rabbit polyclonal anti-cleaved caspase-3 antibody (1:100, Cell Signaling Technology, Frankfurt, Germany).

Techniques: Immunohistochemical staining, Staining

Journal: The American Journal of Pathology

Article Title: Lung remodeling regions in long-term Covid-19 feature basal epithelial cell reprogramming

doi: 10.1016/j.ajpath.2023.02.005

Figure Lengend Snippet: Cell proliferation and apoptosis in lung remodeling regions in Covid-19. A, Immunostaining for Ki-67 plus CD68 with DAPI counterstaining in lung sections from Covid-19 patients and non-disease controls (ND). Scale bar=100 μm; Original magnification, x4 (inset). B , Quantitation of staining for conditions in (A). C , Immunostaining for active caspase-3 with DAPI counterstaining in lung sections for conditions in (A). Scale bar=100 μm. D , Quantitation of staining for conditions in (C). E , Immunostaining for active-caspase-3 plus HT2-280 or CD68 with DAPI counterstaining for conditions in (A). Scale bar=50 μm; Original magnification, x3 (inset). F , Quantitation of staining for conditions in (E). G , Immunostaining for KRT5 plus CXCL17 with DAPI counterstaining for conditions in (A). h, Scale bar=50 μm; Original magnification, x3 (inset). H, Quantitation of staining for conditions in (G). Data are representative of 5 patients and 5 control subjects per staining condition. Values represent mean and s.e.m; * P <0.05 (n=5 patients or subjects per group).

Article Snippet: Immunostaining was performed using the following primary antibodies: rabbit anti-ACE-2 polyclonal Ab (pAb) (ab65863, Abcam), mouse anti-ACE-2 mAb (clone 171606, R&D Systems), rabbit anti-KRT5 pAb (ab53121, Abcam) and mAb (clone EP1601Y, ab52635, Abcam), rabbit anti-AQP3 pAb (ab125219, Abcam), mouse anti-CD68 mAb (clone Kp-1, Sigma-Aldrich), rabbit anti-CD163 mAb (clone D6U1J, Cell Signaling), rabbit anti-CD31 mAb (clone EPR17259, Abcam), rabbit anti-CollagenIV (CollIV) mAb (clone EPR20966, Abcam), mouse anti-SCGB1A1 mAb (clone E-11, Santa Cruz), mouse anti-acetylated Tubulin (clone 6-11B-1, Sigma-Aldrich), mouse anti-MUC5AC mAb (clone 45M1, ThermoFisher Scientific and Santa Cruz Biotechnology), mouse anti-HT2-280 pAb (TB-27AHT2-280, Terrace Biotech), rabbit anti-surfactant protein C (SFTPC) pAb (ab90716, Abcam), rabbit anti-podoplanin (PDPN) mAb (clone EPR7072, Abcam), rabbit anti-MUC5B pAb (ab87276, Abcam), rabbit anti-Ki-67 mAb (clone D2H10, Cell Signaling), rabbit active (cleaved) caspase-3 mAb (clone 5A1E, Cell Signaling), and mouse anti-CXCL17 mAb (clone 422204, R&D Systems).

Techniques: Immunostaining, Quantitation Assay, Staining