Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 with PAI ‐1 si RNA /sh RNA reduces p53 and p21 protein levels, increases Rb phosphorylation, and attenuates bleomycin‐induced L2 cell senescence. Rat ATII (L2) cells were treated with 50 mU /mL bleomycin for 24 h (A & B) and then cultured in bleomycin‐free medium for additional 72 h (B). (C) L2 cell was transfected with PAI ‐1 si RNA or nontarget si RNA ( NT si RNA ). D‐N) PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with bleomycin for 24 h and then cultured in bleomycin‐free medium for additional 24 (D–L) or 72 (M and N) hours. PAI ‐1, serine‐18 phosphorylated p53 (p53 S−18P ), p53, p21, and phosphorylated Rb (ppRb) proteins were determined by Westerns. β‐Actin is used as loading control. D, representative Western blotting pictures; E‐J, semi‐quantified band intensities normalized by β‐actin. (K and L) Immunostaining and quantification of proliferating cell nuclear antigen ( PCNA ). (M and N) SA ‐β‐gal activity revealed by X‐gal staining. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from corresponding NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Cell Culture, Transfection, Stable Transfection, Western Blot, Immunostaining, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Inhibition of PAI ‐1 activity with a small molecule PAI ‐1 inhibitor TM 5275 attenuates bleomycin‐induced L2 cell senescence. L2 cells were treated with 50 mU /mL bleomycin in the presence or absence of 25 μ m of TM 5275 for 24 hours and then cultured in bleomycin‐free medium for additional 72 (A and B) or 24 (C–G) hours. (A and B) SA ‐β‐gal activity was revealed by X‐gal staining; (C–G) Western analyses of the proteins of interested in cell lysates, the band intensities semi‐quantified by ImageJ software, and normalized by β‐actin. α, Significantly different from corresponding saline‐treated cells; β, significantly different from bleomycin‐treated vehicle controls ( P < 0.05, n = 3).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Inhibition, Activity Assay, Cell Culture, Staining, Western Blot, Software

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of PAI ‐1 protein with PAI ‐1 sh RNA attenuates doxorubicin‐induced L2 cell senescence. PAI ‐1 sh RNA or NT sh RNA stably transfected L2 cells were treated with 50 n m of doxorubicin (Dox)/saline for 24 h and cultured in doxorubicin‐free medium for additional 24 h (A–D) or 72 h (E and F). PAI ‐1, p53, p21, and β‐actin in cell lysates were determined by Westerns. A, representative Western blotting pictures; B–D, semi‐quantified band intensities by ImageJ program and normalized by β‐actin. E and F, SA ‐β‐gal activity was revealed by X‐gal staining. α, Significantly different from the corresponding saline‐treated cells; β, significantly different from doxorubicin‐treated NT sh RNA ‐transfected cells; ζ, significantly different from saline‐treated NT sh RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Stable Transfection, Transfection, Cell Culture, Western Blot, Activity Assay, Staining

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockdown of p53 protein with p53 si RNA abrogates PAI ‐1 protein‐mediated L2 cell senescence. (A and B) L2 cells were treated with 1 μg/mL of hPAI ‐1, dissolved in 0.1% BAS , or 0.1% bovine serum albumin ( BSA ) for 72 h. (A) PAI ‐1 mRNA was determined by real‐time PCR ; (B) Proteins of interest were determined by Westerns. (C–I) L2 cells were transfected with p53 si RNA or nontarget si RNA ( NT si RNA ) and then treated with hPAI ‐1 or BSA for 72 h. (C and D) SA ‐β‐gal activity was measured by X‐gal staining. (E–I) Western analyses of proteins of interest; E, representative Western blotting pictures; F–I, semi‐quantified band intensities normalized by β‐actin. α, Significantly different from corresponding 0.1% BSA (solvent) controls; β, significantly different from hPAI ‐1‐treated NT si RNA ‐transfected cells; ζ, significantly different from BSA ‐treated NT si RNA ‐transfected cells ( P < 0.05, n = 3–5).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Staining, Western Blot

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Knockout of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced ATII cell senescence in vivo . (A and B) Double immunostaining of isolated ATII cells with anti‐ PAI ‐1 and anti‐p53 antibodies. (C and D) Double immunostaining of isolated ATII cells with anti‐p21 and anti‐ SPC antibodies. (E and F) SA ‐β‐gal activity in freshly isolated mouse ATII cells was revealed by X‐gal staining. Left panels are representative SA ‐β‐gal staining pictures; right panel is quantitative data. (G–M) Western analyses of the proteins of interest in isolated ATII cells. (N–S) Double‐immunofluorescence staining of mouse lung tissues with PAI ‐1, p53, or p21 and ATII cell marker SPC . Top panels are representative Western blotting pictures, and bottom panels are quantitative data. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice; ζ, significantly different from saline‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–6).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Knock-Out, In Vivo, Double Immunostaining, Isolation, Activity Assay, Staining, Western Blot, Double Immunofluorescence Staining, Marker

Journal: Aging Cell

Article Title: Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

doi: 10.1111/acel.12643

Figure Lengend Snippet: Deletion of the PAI ‐1 gene specifically in ATII cells in mice attenuates bleomycin‐induced lung fibrosis. (A) Body weight changes before and 14 days after bleomycin/saline treatment. (B) The amount of PAI ‐1 protein in BAL fluid measured by ELISA . (C) Trichrome staining of collagen and (D) Sirius red staining of collagen. (E) Hydroxyproline content in mouse lung measured using the Hydroxyproline Assay Kit (Chrondrex, Inc) and expressed as % of hydroxyproline in saline‐treated fl/fl mice. (F–I) Western analyses of procollagen 1α1, procollagen 1α2, and alpha‐smooth muscle actin (α‐ SMA ) in mouse lung tissue. α, Significantly different from same genotype, saline‐treated mice; β, significantly different from bleomycin‐treated PAI ‐1 fl/fl mice ( P < 0.05, n = 3–8).

Article Snippet: To reveal PAI‐1, p53, and p21 proteins in mouse lung tissue, double immunostaining was conducted using formalin‐fixed, paraffin‐embedded tissue slides with antibodies to mouse PAI‐1 (Molecular Innovations, Cat No MA‐33H1F7), p53 (Ancell, Cat No 227‐020), or p21 (Santa Cruz, Cat No SC‐6246) and rabbit polyclonal anti‐mouse proSP‐C antibody (Millipore, Cat No AB3786), following the protocol as we have described previously (El‐Deiry et al ., ).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Hydroxyproline Assay, Western Blot

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: D1r downregulation in adolescence is associated with complement C3 and microglial engulfment in males, but not the females. Triple-label immunohistochemistry for D1r, C3, and Iba1 was performed and protein expression levels for each target were normalized for z-stack size. Because male and female tissue was processed at different times and required different image acquisition parameters, neither data nor images can be directly compared between the sexes. Representative images precede histograms; scale bars are equal to 20 µm. Males: a D1r levels transiently increased at P30 (Table ; b C3 levels decreased after P30 (Table ; c Iba1 levels decreased after P30 (Table . d Merged male images. Females: e D1r levels decreased after P20 (Table f C3 levels transiently increased at P38 (Table ; g Iba1 levels decreased after P20 (Table . h Merged female images. Images were then analyzed using a volumetric reconstruction of Iba1 (i.e., microglia) as a mask to determine the volume of D1r, C3, or colocalized C3-D1r physically associated with (either in contact with or inside) microglia. Masked volumes were normalized for total protein of the target and total Iba1 (see Methods). i Representative 2D triple-label immunohistochemistry (scale bar 20 µm), with enlarged 3D representation (scale bar 3 µm) of the selected area demonstrating (left-right) D1r, C3, and C3-D1r contacted by/internal to the microglia. Open arrow head indicates a region of the microglia where both D1r and C3 are located, but not colocalized, demonstrating the sensitivity of colocalization analyses. Closed arrow head indicates a region of the microglia where D1r and C3 colocalize (C3-D1r). Males: j D1rs are maximally contacted by microglia (irrespective of changing D1r and Iba1 levels) at P30 (Table ). k C3 contact by microglia does not change over time (Table ), and L C3-D1r contact is high between P30-P38 (Table ). Females: m D1rs are maximally contacted by microglia at P54, not at P20, prior to their elimination (Table ). n C3 contact is also increased at P54 (Table ), while o C3-D1r contact does not change over development (Table )

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: D1r downregulation in adolescence is associated with complement C3 and microglial lysosomal degradation in the male, but not the female, NAc. To assess microglial phagocytic activity and whether D1r, C3, or C3-D1r were localized in microglial CD68+ lysosomes, immunohistochemistry for CD68, D1r, and C3 was performed. a Representative 2D triple-label immunohistochemistry (scale bar 20 µm), with enlarged 3D representation (scale bar 2 µm) of the selected area demonstrating (left-right) D1r, C3, and C3-D1r within CD68+ lysosomes. Closed arrow head indicates a lysosome where D1r and C3 colocalize (C3-D1r). Males: c CD68 levels are high from P30 to 38 (Table ). b D1r content in CD68+ lysosomes is highest at P30 (Table ), d C3 content in lysosomes does not change over development (Table ), and e C3-D1r content in lysosomes is transiently elevated at P30 (Table ). Females: f CD68 levels are transiently elevated at P30 (Table ). g D1r content in CD68+ lysosomes is high at both P30 and P54 (Table ), h C3 content in lysosomes is highest at P20 and P54 (Table ), and i C3-D1r content in lysosomes does not change over development (Table ). For all experiments, n = 4 animals/sex/group. Data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Activity Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R interactions mediate developmentally-typical D1r elimination in vivo in males, but not females. Neutrophil inhibitor factor (NIF), a peptide that binds specifically to the CD11b subunit of C3 receptors (C3Rs), was assessed for its efficacy in reducing microglial phagocytic activity ex vivo. a In microglia isolated from whole brain, 60 ng, but not 120 ng NIF inhibited microglial phagocytosis of fluorescent beads (Table ). Representative images precede histograms; scale bar equals 20 µm. n = 4, with 2 replications/condition. b Microglia were isolated from whole brain and incubated with 60 ng NIF, and then immunocytochemically assessed for NIF and CD11b at 30, 60, and 120 min. Closed arrow heads indicate NIF (green) immunoreactivity. Scale bar equals 10 µm. There was no change in c NIF or (Table ) d CD11b (Table ) immunoreactivity over 2 h, suggesting that NIF was not causing the degradation of its receptor. n = 3; 2 replications/condition. To determine if developmental D1r downregulation requires C3–C3R interactions, NIF or Vehicle was microinjected in the NAc at e P30 in males and g P22 in females (both represent sex-specific ages prior to D1r downregulation), and then tissue was assessed at P38 and P30, respectively, an age at which D1rs should be developmentally downregulated. f In males, NIF-treated hemispheres exhibited significantly more (~25%) D1r immunoreactivity than within-animal vehicle-treated hemispheres (Table ). h In females, NIF-treated hemispheres exhibited the same D1r immunoreactivity as within-animal vehicle-treated hemispheres (Table ). Representative images precede histograms; scale bars equal 100 µm. n = 4/sex with counterbalanced injections. For ex vivo experiments, data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. For in vivo experiments, D1r data from 4–7 different sections per animal were averaged and then calculated as a percentage of within-animal vehicle control levels. Data were analyzed with 2-tailed one-sample t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( a – d ) and one-sample t -tests from 100 ( f , h ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: In Vivo, Activity Assay, Ex Vivo, Isolation, Incubation

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Sex-specific social behavior patterns over adolescence require C3–C3R interactions. a Experimental design: separate cohorts of experimental animals ( n = 8/sex/age) were single-housed for 24 h prior to test, after which time a novel age-matched and sex-matched conspecific was introduced into their home cage. Ten minutes of interactions were recorded, and later an experimenter blinded to the conditions coded the interactions for either total play or social exploration (i.e., non-play social behavior). Only behaviors initiated by the experimental animal were considered. Males: b Male social play behavior transiently increased at P30 (Table ), c with no changes in social exploration over development (Table ). Females: d Female social play behavior did not change over development (Table ), e while social exploration peaked from P30 to 38 (Table ). f To determine if C3–C3R interactions regulating D1r levels between P30 and 38 in males is required for the decline in social play behavior observed at this time, NIF or vehicle was injected bilaterally in P30 males or P22 females ( n = 10/sex/treatment). Animals were then single housed for 24 h at P37 or P29, and assessed for social behavior as described at P38 and P30, respectively. Males: g Interrupting C3–C3R interactions at P30 increased social play behavior at P38 (Table ), h without affecting social exploration (Table ). Females: i Female social play behavior, while at a low basal level, also modestly increased at P30 after P22 NIF injection (Table , j with no changes in social exploration (Table ). Data were analyzed either with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons or with two-tailed unpaired t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( b – e ) and unpaired t -tests ( g – j ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R regulation of D1rs regulates male, but not female, social play behavior. To determine if disrupting C3–C3R interactions via NIF increases play in a D1r-dependent manner, we utilized siRNA against rat D1r (200 µM) or scRNA controls. a P30 males were co-injected with NIF + D1r siRNA in one hemisphere and NIF + scRNA in the contralateral hemisphere, and then D1r immunoreactivity was assessed at P38. b In the presence of NIF, D1r siRNA was capable of decreasing D1r levels relative to NIF + scRNA within-animal control hemispheres (~20%; Table ). c P22 females were injected with NIF + D1r siRNA in one hemisphere and NIF + scRNA in the contralateral hemisphere, and then D1r immunoreactivity was assessed at P30. d In the presence of NIF, D1r siRNA was capable of decreasing D1r levels relative to NIF + scRNA within-animal control hemispheres (~30%; Table ). Representative images precede histograms; scale bar equals 100 µm. n = 3/sex. e Experimental design: P30 males and P22 females were bilaterally microinjected into the NAc with either Vehicle + scRNA (Veh sc), Vehicle + D1r siRNA (Veh si), NIF + scRNA (NIF sc), or NIF + D1r siRNA (NIF si). At P37 and P22, animals were single-housed for 24 h, and then a novel age- and sex-matched conspecific was introduced into their home cage for 10 min social behavior tests ( n = 9–10/sex/treatment). Males: f NIF + scRNA increased social play behavior in males, which was eliminated by D1r siRNA (NIF si; Table ). g No treatment changed social exploration behavior (Table ). Females: h NIF + scRNA increased social play behavior in females, which was not affected by D1r siRNA (NIF si; Table ). i No treatment changed social exploration behavior (Table ). Immunohistochemical data were calculated as in Fig. and analyzed with two-tailed one-sample t -tests. Behavioral data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. Histograms portray the mean ± SEM with individual data points overlaid. Significant one-sample t -tests from 100 ( b , d ) and post-hoc Holm-Sidak t -tests ( f – i ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques: Injection, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 1

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques:

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 2

Article Snippet: Tissue was permeabilized for 1 h with 0.3% Triton-x100 in PBS and then blocked in 10% normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PBS for 1 h. Primary antibodies were applied sequentially for overnight incubations in 5% normal donkey serum in PBS at 4 °C: either anti-goat C3 (MP Biomedicals #0855713; Santa Ana, CA; 1:200), anti-mouse D1r (Novus Biologicals #NB110–60017; Littleton, CO; 1:750), anti-rabbit Iba1 (Wako Chemicals #019–19741; Richmond, VA; 1:1500) or anti-rabbit CD68 (Abcam #ab125212; Cambridge, MA; 1:5000), anti-goat C3, anti-mouse D1r.

Techniques:

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Tissue plasminogen activator (t-PA) is present on the outer leaflet of the plasma membrane of stimulated chromaffin cells. ( a ) Dual labeling of cell surface t-PA and exocytotic sites. Cells were stimulated with nicotine 20 µM in the presence of anti-t-PA and anti-DBH antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor ® 488 and Alexa Fluor ® 551, respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 µM. ( b ) The t-PA labeling on the surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine without (S) or after EGTA wash (S + EGTA). Anti-t-PA antibodies were revealed with Alexa Fluor ® 488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. Confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 µM. ( c ) Semi-quantitative analysis of t-PA labeling on the cell surface of chromaffin cells in the resting condition (R), stimulated with 20 µM nicotine (S) without (Control) or after EGTA wash (S + EGTA). The t-PA labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (*** = p < 0.001, ** = p < 0.01). Three experiments were done on independent cell cultures and pooled ( n = 36 and 65 control cells, 40 and 75 EGTA-treated cells for resting and stimulated conditions, respectively). ( d ) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R) or stimulated with nicotine 20 µM (S) were analyzed by western blot and revealed with anti-t-PA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Incubation, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

doi: 10.3390/cells9092059

Figure Lengend Snippet: Membrane topography of AnxA2, t-PA and exocytotic sites after immunogold labeling of the outer face of the plasma membrane sheets prepared from stimulated chromaffin cells. ( a ) Plasma membrane sheets were prepared from bovine chromaffin cells stimulated by nicotine for 5 min. To label DBH, AnxA2 and t-PA exposed at the surface of cells undergoing exocytosis, anti-DBH, anti-AnxA2 and anti-t-PA antibodies were added during stimulation. Membrane sheets were labeled with anti-mouse antibodies coupled to 10 nm gold particles to detect DBH antibodies revealing exocytotic sites (red circle) and rabbit antibodies coupled to 15 nm gold particles to label AnxA2 or t-PA (green circle). ( b ) The histogram represents the relative distribution of 15 nm gold particles as a function of their distance from the granule membrane once inserted in the plasma membrane (blue line). The distance was measured and the number of particles was counted manually with Photoshop. Three experiments were done on independent cell cultures ( c ). Double staining experiment for t-PA (10 nm gold particles) and AnxA2 (15 nm gold particles) were performed with the same protocol. Scale bar: 100 nm. ( d ) The histogram represents the relative distribution of 10 nm gold particles (t-PA) as a function of their distance from 15 nm gold particles (AnxA2). The distance was measured and the number of particles was counted manually with Photoshop. Two experiments were done on two independent cell cultures.

Article Snippet: Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA).

Techniques: Labeling, Double Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Urine complement activation fragments are increased in patients with kidney injury after cardiac surgery

doi: 10.1152/ajprenal.00130.2019

Figure Lengend Snippet: The alternative pathway of complement is activated in mice after kidney ischemia-reperfusion (IR). C57BL/6 mice were subjected to 24 min of kidney ischemia or to sham surgery. A: kidneys were examined by immunofluorescence microscopy for C3b (green) and C4 (red), and nuclei were stained with DAPI (blue). C4 was seen in the glomeruli (arrowheads), and C3b was seen along the tubules (arrows) of sham-treated animals. C3b and C4 deposition was more intense in mice after ischemia and 24 h of reperfusion (T24 IR). C3 staining was still not seen in the glomeruli, however, and C4 staining was not seen in the tubulointerstitium. Control staining with isotype-matched antibodies was negative. Original magnification ×200. Scale bar = 50 μm. B: urine Ba fragments were measured by ELISA in urine samples collected before IR (pre) or after 3 h of reperfusion (3 h). Levels were significantly higher after IR. ***P < 0.001. C: Western blot analysis of urine for C4 showed that levels of intact C4 as well as C4 fragments were higher in samples collected after 3 h of reperfusion. WT, wild-type.

Article Snippet: The following primary antibodies were used in these studies: FITC-conjugated goat IgG to mouse complement C3 (MP Biomedicals, Santa Ana, CA), a monoclonal antibody (mAb) that only recognizes the iC3b and C3d activation fragments of C3 (mAb 3d29) ( 30 ), an antibody to C4 (Hycult Biotech, Uden, The Netherlands), goat anti-human properdin factor B (DiaSorin/Bio-Rad, Hercules, CA), and a biotinylated mAb to mouse factor B that binds to the Ba fragment ( 29 ).

Techniques: Immunofluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Western Blot