alexa fluor 488  (Thermo Fisher)

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: TRPV1 in Salivary Gland Epithelial Cells Is Not Involved in Salivary Secretion via Transcellular Pathway

doi: 10.4196/kjpp.2014.18.6.525

Figure Lengend Snippet: Distribution of TRPV1 in salivary gland epithelial cells (SGEC). Representative immunofluorescence labeling of TRPV1 in mSMG (A), hSMG (B), and HSG cells (C). SMGs sections and HSG were immunostained with anti-TRPV1 antibody and AQP5 antibody, then incubated with Alexa Fluor-linked anti-rabbit IgG (red) and Alexa Fluor-linked anti-goat IgG (green). Nuclei (blue) were labeled with 4,6-diamidino-2-phenylindole. (A) TRPV1were mainly detected in apical membrane in acinar cells and in basolateral membrane in ductal cells. AQP5 was used as a marker for acinar cells that was exclusively expressed in the apical membrane of acinar cells (red color). The merged orange color indicates that expression of TRPV1 at the apical membrane in acinar cells. (B) In hSMG, higher expression levels of TRPV1 was observed in ductal cells compared to acinar cells. (C) In HSG cells, TRPV1 was widespread in the cytoplasm. Scale bar indicates 20 µm. The results are representative of four independent experiments.

Article Snippet: Reagents Capsaicin, carbachol, and pilocarpine (Sigma Aldrich, St.Louis, MO, USA), Fura-2/AM (Molecular Probes, Eugene, OR, USA), TRPV1 antibody (Abcam, Cambridge, UK; Santa Cruz Biotechnology; Santa Cruz, CA), AQP5 antibody (Abcam, Santa Cruz Biotechnology), Alexa Fluor® 594 donkey anti-rabbit IgG and Alexa Fluor® 488 donkey anti-goat IgG (Invitrogen Corporation, Carlsbad, CA, USA), and normal donkey serum (Jackson ImmunonoResearch, West Grove, PA, USA), were used in this study.

Techniques: Immunofluorescence, Labeling, Incubation, Marker, Expressing

Journal: Scientific Reports

Article Title: TRSP is dispensable for the Plasmodium pre-erythrocytic phase

doi: 10.1038/s41598-018-33398-8

Figure Lengend Snippet: TRSP - sporozoite entry in hepatocytes. ( A ) Representative immunofluorescence image of an anti-CSP double staining using control sporozoites in an in vitro entry assay. Intracellular sporozoites (In) are labelled in green whilst extracellular sporozoites (Out) appear in red and green. DAPI-stained nuclei appear in blue. The image on the right represents a magnified section of the image on the left. ( B – E ) Comparison of the number of sporozoites per field stained with anti-CSP antibodies and Alexa Fluor 488-conjugated ( B ) or Alexa Fluor 568-conjugated ( C ) secondary antibodies; intracellular sporozoites ( D ), corresponding to the number of Alexa Fluor 568 + parasites subtracted from the number of Alexa Fluor 488 + parasites; and nuclei ( E ) in a representative anti-CSP double staining, determined manually or by automated analysis. ( F ) Percentage of HepG2 cells infected by control and TRSP - I21 (graph on the left) or I26 (graph on the right) sporozoites, in 25 fields from each replicate well. ( G ) Partial entry events were defined as Alexa 488 + sporozoites stained with Alexa 568 secondary antibody up to roughly 50% of its length in an anti-CSP double staining. A partial entry event of control sporozoites in an immunofluorescence image is identified with a white arrowhead. DAPI-stained nuclei appear in blue ( H ) Percentage of cells exhibiting partial entry events using TRSP - I21 (graph on the left) or I26 (graph on the right) sporozoites. Ten fields of a representative assay were analysed and events were counted manually. ( I ) Percentage of sporozoites partially inserted into the hepatocyte membrane using TRSP - I21 (graph on the left) or I26 (graph on the right) sporozoites. This parameter represents the number of partial entry events out of the total number of intracellular parasites in a field, represented as a percentage. Means + SD ( F ) or means ± SD ( H,I ) are represented. Four independent experiments per clone were performed. Statistical analysis was performed using the Mann Whitney test: n.s., non-significant.

Article Snippet: Finally, the goat α-mouse Alexa Fluor 488 (1:500; Invitrogen) antibody was used to stain all parasites.

Techniques: Immunofluorescence, Double Staining, In Vitro, Staining, Infection, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

doi: 10.1093/nar/gky403

Figure Lengend Snippet: FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by Alexa fluor 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P

Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

Techniques: Mutagenesis, Fluorescence, Recombinant, Western Blot, Transfection, Plasmid Preparation, Expressing, Labeling, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

doi: 10.1093/nar/gky403

Figure Lengend Snippet: FANCJ-R707C fails to suppress single-stranded DNA or Rad51 persistence in cells exposed to CisPt. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to CisPt (1 μM) ( A, B ) or APH (200 nM) ( C, D ). Note that for BrdU staining, cells were pre-labeled with BrdU prior to drug exposure as described in Materials and Methods. (A, C) Immunofluorescent BrdU foci (A) or Rad51 foci (C) were detected by Alexa fluor 488 and are shown along with DAPI, or merged (DAPI and Alexa fluor 488). (B, D) Quantitative analyses of BrdU foci (B) or Rad51 foci (D) are shown with S.D. indicated by error bars.

Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

Techniques: Transfection, Plasmid Preparation, BrdU Staining, Labeling