Journal: BMC Neuroscience

Article Title: Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer’s disease

doi: 10.1186/s12868-016-0288-1

Figure Lengend Snippet: Effect of PICALM siRNA on clathrin-mediated endocytosis. H4 cells were incubated with either control siRNA (GFP siRNA) or siRNA targeting PICALM (PICALM siRNA) prior to incubation with 100 nM Alexa488-Tf for 5, 15 or 30 minutes then trypsinised and analysed by flow cytometry. Mean fluorescent intensity data for the uptake of Alexa488-Tf are expressed as fold change over untreated control cells (0 min Alexa488-Tf) and are mean ± SEM, ***p < 0.001, **p < 0.01 significantly different to GFP siRNA with an unpaired Student’s t test, n = 3

Article Snippet: Cells were then washed twice with Opti-MEM at room temperature and incubated for 0–30 min with Opti-MEM containing 100 nM Alexa488-labelled transferrin (Alexa488-Tf; Life Technologies).

Techniques: Incubation, Flow Cytometry

Journal: BMC Neuroscience

Article Title: Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer’s disease

doi: 10.1186/s12868-016-0288-1

Figure Lengend Snippet: Effects of clathrin siRNA on clathrin-mediated endocytosis. H4 cells were incubated with media, control siRNA (GFP siRNA) or siRNA targeting clathrin heavy chain (Clathrin siRNA) for 48 h. a Representative immunoblot of clathrin expression and b following densitometric analysis. Data are presented as a percentage of the untreated media control after normalisation to house-keeping protein levels and are mean ± SEM, n = 3, *p < 0.05 significantly different to GFP siRNA with an unpaired Student’s t test. c Clathrin mRNA expression levels using quantitative PCR, normalised to the housekeeping gene 18S and relative to the untreated cells. Data are presented as mean ± SEM, n = 6, **p < 0.01 significantly different to GFP siRNA with Mann–Whitney-U. d H4 cells were incubated with 100 nM Alexa488-Tf for 5, 15 or 30, minutes then trypsinised and analysed by flow cytometry. Mean fluorescent intensity (MFI) data are expressed as fold change over untreated control cells (0 min Alexa488-Tf) and are mean ± SEM *p < 0.05,**p < 0.01 significantly different to GFP siRNA with an unpaired Student’s t test, n = 4–5

Article Snippet: Cells were then washed twice with Opti-MEM at room temperature and incubated for 0–30 min with Opti-MEM containing 100 nM Alexa488-labelled transferrin (Alexa488-Tf; Life Technologies).

Techniques: Incubation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Flow Cytometry

Journal: Scientific Reports

Article Title: Localization and density of Porphyromonas gingivalis and Tannerella forsythia in gingival and subgingival granulation tissues affected by chronic or aggressive periodontitis

doi: 10.1038/s41598-018-27766-7

Figure Lengend Snippet: Western blot analysis for reactivity of the anti-PG and anti-TF antibodies. Western blot analysis for reactivity of the anti-PG antibody ( a ; upper column), the anti-TF antibody ( b ; middle column), and the PBS control without primary antibody ( c ; lower column) was performed using sonicated whole-cell lysate. M; marker of molecular weight (molecular mass in kiloDaltons), Lane 1; PG (ATCC 33277), lane 2; TF (ATCC 43037), lane 3; Aggregatibacter actinomycetemcomitans (ATCC 43718); lane 4, Fusobacterium nucleatum (ATCC 25586), lane 5; Prevotella intermedia (ATCC 25611), lane 6; Prevotella nigrescens (ATCC 33563), lane 7; Bacteroides fragilis (ATCC 25285), and lane 8; Bacteroides vulgatus (ATCC 25285). Both the anti-PG and anti-TF antibodies exhibited a ladder pattern of positive bands ranging from 31 to 76 kDa and from 38 to 225 kDa only on the PG and TF lanes ( a , b ), respectively. No positive bands were observed in the other lanes or in the PBS control ( c ).

Article Snippet: Four strains of PG (ATCC 53978, ATCC 33277, BAA-1703, and A7A128) and four strains of TF (ATCC 43037, and 3 clinical isolates) were cultured on Tripticase soy agar , plates containing defibrinated horse blood, hemin, and vitamin-K. For TF strain cultures, 15 μl of 20 mg/ml N-acetyl muramic acid solution containing 100 μm sterilised paper disc per Tripticase soy agar plate was added.

Techniques: Western Blot, Control, Sonication, Marker, Molecular Weight

Journal: PLoS ONE

Article Title: Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

doi: 10.1371/journal.pone.0032794

Figure Lengend Snippet: Activation of monocytes in the circulation is characterized by increased activation of the inflammatory toll-like receptor-2 (TLR2)/NFκB signaling pathway. Activation of this pathway will elicit pro-inflammatory cytokine release (e.g. tumor necrosis factor α (TNFα)), mitochondrial and intracellular ROS production and impairment of the insulin sensitivity at the molecular level. More mitochondrial ROS production is associated with an increased expression of the mitochondrial antioxidant superoxide dismutase-2 ( SOD2 ), suggesting that SOD2 only acts as mitochondrial stress marker (indicated as dotted line). The formed ROS will not only induce the IKK complex phosphorylation and thereby the translocation of NFκB but will also lead to inhibition of the insulin receptor signaling pathway. Interestingly, interleukin-1 receptor-associated kinase-3 (IRAK3) and miR-146b-5p are located upstream of the transcription factor NFκB and negatively regulates TLR signaling. IRAK3 prevents the dissociation of IRAK1 and IRAK4 from MyD88 and formation of IRAK-TRAF6 complexes. miR-146b-5p, on the other hand, inhibits the translation of IRAK1 and TRAF6. Flow of the pathway at the protein interaction level is indicated by black arrows. Blunted arrows indicate inhibition. Phosphorylation is indicated by a circled P.

Article Snippet: NFκB p65 DNA binding activity was assessed on isolated nuclear extracts of transfected THP-1 monocytes by ELISA using the TransAM NFκB p65 transcription factor assay kit according to the manufacturer's protocol (Active Motif, La Hulpe, Belgium).

Techniques: Activation Assay, Expressing, Marker, Phospho-proteomics, Translocation Assay, Inhibition

Journal: PLoS ONE

Article Title: Decrease of miR-146b-5p in Monocytes during Obesity Is Associated with Loss of the Anti-Inflammatory but Not Insulin Signaling Action of Adiponectin

doi: 10.1371/journal.pone.0032794

Figure Lengend Snippet: ( A ) IRAK1 and TRAF6 protein levels, ( B ) NFκB p65 DNA binding activity and TNFα RNA levels, ( C ) IRAK3 RNA and protein levels, ( D ) m- and iROS production and ( E ) IRS1 RNA and protein levels in THP-1 cells transfected with miR-146b-5p inhibitor as determined by qRT-PCR, ELISA, Western blotting and flow cytometry. Data are expressed as means ± SEM. n = 6. * P <0.05 and ** P <0.01 compared with THP-1 cells transfected with negative control miRNA. Abbreviations: iROS, intracellular ROS; mROS, mitochondrial ROS; ROS, reactive oxygen species.

Article Snippet: NFκB p65 DNA binding activity was assessed on isolated nuclear extracts of transfected THP-1 monocytes by ELISA using the TransAM NFκB p65 transcription factor assay kit according to the manufacturer's protocol (Active Motif, La Hulpe, Belgium).

Techniques: Binding Assay, Activity Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Negative Control

Journal: The journals of gerontology. Series A, Biological sciences and medical sciences

Article Title: PGC-1beta down-regulation is associated with reduced ERRalpha activity and MCAD expression in skeletal muscle of senescence-accelerated mice.

doi: 10.1093/gerona/61.8.773

Figure Lengend Snippet: Figure 3. Increased expression of chicken ovalbumin upstream promoter transcription factor II (COUP-TF II) protein levels in skeletal muscle of senescence-accelerated prone mice (SAM-P8). Nuclear extracts from soleus skeletal muscle from senescence-accelerated resistant mice (SAM-R1) and SAM-P8 were subjected to immunoblot analysis as described in Methods. Representative immunoblots using antiperoxisome proliferator-activated re- ceptor (PPAR)a, anti-PPARb/d, COUP-TF II, and b-tubulin antibodies are shown. Autoradiograph data are representative of three animals.

Article Snippet: Samples were analyzed using antibodies that recognize PPARa (Santa Cruz Biotechnology, Santa Cruz, CA), PPARb/d (gift from Dr. Walter Wahli, University of Lausanne, Switzerland), chicken ovalbumin upstream promoter transcription factor II (COUP-TF II; Santa Cruz Biotechnology), D ow nloaded from https://academ ic.oup.com /biom edgerontology/article/61/8/773/589261 by guest on 03 August 2024 and b-tubulin (Sigma) by western blot analysis.

Techniques: Expressing, Western Blot, Autoradiography

Journal: The journals of gerontology. Series A, Biological sciences and medical sciences

Article Title: PGC-1beta down-regulation is associated with reduced ERRalpha activity and MCAD expression in skeletal muscle of senescence-accelerated mice.

doi: 10.1093/gerona/61.8.773

Figure Lengend Snippet: Figure 4. Binding of cardiac nuclear proteins to a peroxisome proliferator-response element (PPRE) probe is reduced in nuclear extracts from skeletal muscle of senescence-accelerated prone mice (SAM-P8). A, Autoradiograph of electrophoretic mobility shift assay (EMSA) performed with a 32P-labeled PPRE nucleotide and crude nuclear protein extract (NE) shows two specific complexes (I and II), based on competition with a molar excess of unlabeled probe. Autoradiograph of EMSA performed with a 32P-labeled PPRE nucleotide and NE from skeletal muscle of senescence-accelerated resistant mice (SAM-R1) and SAM-P8. B, Identification of chicken ovalbumin upstream promoter transcription factor II (COUP-TF II) in skeletal muscle nuclear protein complexes by antibody recognition experiments. Autoradiograph depicted represents EMSA antibody recognition studies performed with a PPRE probe and anti-COUP-TF II antibody. C, Autoradiograph of EMSA performed with a 32P-labeled nuclear factor-jB (NF-jB) nucleotide and NEs. One specific complex (I), based on competition with a molar excess of unlabeled probe, is shown. Autoradiograph data are representative of three separate experiments.

Article Snippet: Samples were analyzed using antibodies that recognize PPARa (Santa Cruz Biotechnology, Santa Cruz, CA), PPARb/d (gift from Dr. Walter Wahli, University of Lausanne, Switzerland), chicken ovalbumin upstream promoter transcription factor II (COUP-TF II; Santa Cruz Biotechnology), D ow nloaded from https://academ ic.oup.com /biom edgerontology/article/61/8/773/589261 by guest on 03 August 2024 and b-tubulin (Sigma) by western blot analysis.

Techniques: Binding Assay, Autoradiography, Electrophoretic Mobility Shift Assay, Labeling

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 is upregulated by NF‐κB and YY1. (A) The predicted NF‐κB and YY1 binding sites on LINC01578 promoter. NF‐κB and YY1 bind sites were at −438 and −4 positions relative to the transcription start site, respectively. (B) ChIP assays were performed in DLD‐1 cells to measure the binding of NF‐κB and YY1 on LINC01578 promoter. A distant region without NF‐κB and YY1 binding sites was used as NC (P3). (C) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with PBS or 10 ng·mL −1 TNF‐α for 24 h were detected by western blot. (D) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with DMSO or 5 µ m JSH‐23 for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with DMSO or 5 µ m JSH‐23 for 24 h were detected by western blot. (E) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and p65 overexpression vector. p65 overexpression efficiencies were detected by western blot. (F) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against p65. p65 knockdown efficiencies were detected by western blot. (G) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and YY1 overexpression vector. YY1 overexpression efficiencies were detected by western blot. (H) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against YY1. YY1 knockdown efficiencies were detected by western blot. (I) LINC01578 expression in DLD‐1 cells treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. (J) LINC01578 expression in DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 for 24 h. (K) LINC01578 expression in DLD‐1 cells transfected with p65 overexpression vector. (L) LINC01578 expression in DLD‐1 cells transfected with siRNAs against p65. (M) LINC01578 expression in DLD‐1 cells transfected with YY1 overexpression vector. (N) LINC01578 expression in DLD‐1 cells transfected with siRNAs against YY1. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant, by one‐way ANOVA followed by Dunnett's multiple comparisons test (B) or Student's t ‐test (C‐N).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Binding Assay, Luciferase, Transfection, Western Blot, Over Expression, Plasmid Preparation, Knockdown, Expressing

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 activates NF‐κB signaling. (A) p50 IHC staining of liver metastatic tumors derived from LINC01578‐overexpressed and control DLD‐1 cells. Scale bars, 50 µm. (B) p50 IHC staining of liver metastatic tumors derived from LINC01578‐depleted and control LoVo cells. Scale bars, 50 µm. (C) Nuclear p50 and p65 levels in LINC01578‐overexpressed and control DLD‐1 cells were detected by western blot. (D) Nuclear p50 and p65 levels in LINC01578‐depleted and control LoVo cells were detected by western blot. (E) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐overexpressed and control DLD‐1 cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (F) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐depleted and control LoVo cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (G) p50 and p65 activation in nuclear extracts from LINC01578‐overexpressed and control DLD‐1 cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. (H) p50 and p65 activation in nuclear extracts from LINC01578‐depleted and control LoVo cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. Data are shown as mean ± SD based on n = 6 mice in each group (A,B) or three independent experiments (C–H). * P < 0.05, ** P < 0.01 by the Mann–Whitney test (A), Kruskal–Wallis test followed by Dunn's multiple comparisons test (B), or one‐way ANOVA followed by Dunnett's multiple comparisons test (E–H).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Immunohistochemistry, Derivative Assay, Control, Western Blot, Luciferase, Binding Assay, Transfection, Activity Assay, Activation Assay, Transcription Factor Assay, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 forms a positive feedback loop with NF‐κB/YY1. (A) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector were determined by qRT‐PCR and western blot. (B) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 were determined by qRT‐PCR and western blot. (C) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (D) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (E) A schematic model of positive feedback loop between LINC01578 and NF‐κB/YY1. (F) Luciferase reporter assays for LINC01578‐overexpressed and control DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (G) Luciferase reporter assays for LINC01578‐depleted and control LoVo cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (H) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (I) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. (J) ChIP assays using YY1 antibody were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (K) ChIP assays using YY1 antibody were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant, by Student's t ‐test (A,C,F,H,J), or one‐way ANOVA followed by Dunnett's multiple comparisons test (B,D,G,I,K).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Control

Journal: Cell death discovery

Article Title: Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

doi: 10.1038/cddiscovery.2015.51

Figure Lengend Snippet: Figure 3. FVII, but not TF, increases PAR2 expression and p-ERK1/2 levels in dose-dependent and time-dependent manners. Quiescent monolayers of Hep3B (a) and HepG2 (b) cells (1 × 105) were treated with a control serum-free medium or medium supplemented with recombinant FVIIa (NovoSeven RT) for series of concentrations (10, 20, 40, 60, 80, 100, 150 and 200 ng/ml). After 6 h, cells were harvested and detected for TF, FVII, PAR2, p-ERK1/2 and β-actin by western blot analysis. The levels of PAR2 and p-ERK1/2 were gradually increased both in Hep3B and HepG2 cells. (c) The levels of PAR2 and p-ERK1/2 were significantly reduced by knockdown of FVII using siRNA both in Hep3B and HepG2 cells. (d) Hep3B cells were treated with a control serum-free medium or medium supplemented with TF (Merck) for series of concentrations (10, 20, 40, 60, 80, 100, 150 and 200 ng/ml). After 6 h, cells were harvested and detected for TF, FVII, PAR2, p-ERK1/2 and β-actin by western blot analysis. The levels of PAR2 and p-ERK1/2 were not gradually increased by increasing concentration of TF. (e) Hep3B cells were treated with medium supplemented with FVIIa for a time-series analysis (0, 1, 3, 6, 12 and 24 h). The levels of PAR2, p-ERK1/2 but not TF were gradually increased in a time-dependent manner.

Article Snippet: Recombinant human TF and FVII were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control, Recombinant, Western Blot, Knockdown, Concentration Assay

Journal: Molecular Metabolism

Article Title: Aregs-IGFBP3-mediated SMC-like cells apoptosis impairs beige adipocytes formation in aged mice

doi: 10.1016/j.molmet.2025.102125

Figure Lengend Snippet: Aging induces an elevation in Aregs and a reduction in SMC-like cells among ASPCs following cold exposure . (A) UMAP plot showing the clustering distribution of 58,714 cells from young and aged mice sWAT. (B) Dot plot displaying the expression levels and proportions of selected marker genes across different cell types. The size of the dot represents the percentage of cells expressing the gene, while the color intensity reflects the average gene expression level. (C) Stacked bar chart showing the proportion of cell types in sWAT at different time points (TN, 3 days, and 14 days post-cold exposure). (D) Ucp1 and Cd142 mRNA expression levels analyzed by qPCR in sWAT (n = 11). (E) Immunoblots for CD142, UCP1, TOMM20 and GAPDH in sWAT (n = 3). (F) Quantification of immunoblots of CD142, UCP1 and TOMM20, protein levels are normalized to GAPDH. (G) Immunohistochemistry fluorescence for UCP1 (red) and CD142 (green) (scale bar = 50 μm or 10 μm). Data represent mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After washing with PBST, sections were incubated for 3 h at RT in the dark with anti-goat FITC secondary antibody (1:400, Proteintech, SA00003-3) for CD142, anti-rabbit CoraLite594 secondary antibody (1:400, Proteintech, SA00013-8) for UCP1, and DAPI (Invitrogen, D1306).

Techniques: Expressing, Marker, Gene Expression, Western Blot, Immunohistochemistry, Fluorescence

Journal: Molecular Metabolism

Article Title: Aregs-IGFBP3-mediated SMC-like cells apoptosis impairs beige adipocytes formation in aged mice

doi: 10.1016/j.molmet.2025.102125

Figure Lengend Snippet: Aregs and IGFBP3 inhibit beige adipocyte formation. (A–E) All five groups of ASPCs underwent the same browning differentiation protocol. (A) Ucp1 mRNA expression levels analyzed by qPCR. (B) Immunoblots for UCP1, TOMM20 and GAPDH. (C,D) Quantification of immunoblots of UCP1 and TOMM20, protein levels are normalized to GAPDH. (E) Immunofluorescence staining for CD142 and Mitotracker for mitochondria (scale bar = 20 μm). Data represent mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After washing with PBST, sections were incubated for 3 h at RT in the dark with anti-goat FITC secondary antibody (1:400, Proteintech, SA00003-3) for CD142, anti-rabbit CoraLite594 secondary antibody (1:400, Proteintech, SA00013-8) for UCP1, and DAPI (Invitrogen, D1306).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining