Journal: The Journal of Neuroscience

Article Title: Linking Cell Surface Receptors to Microtubules: Tubulin Folding Cofactor D Mediates Dscam Functions during Neuronal Morphogenesis

doi: 10.1523/JNEUROSCI.0973-14.2015

Figure Lengend Snippet: Absence of tubulin labeling in TBCD1 PNs. A–D, UAS-βTub56D-myc was expressed in WT (A, C) and TBCD1 (B, D) DL1 PNs. PN morphologies are labeled in green, red shows βTub56D-myc staining, and blue shows anti-DN-cadherin staining. A, C, βTub56D-myc is present throughout the axons and dendrites of WT DL1 PNs. B, D, No βTub56D-myc labeling detected in TBCD1 DL1 PNs. E–P, Representative images of dendrite (E–J) and axon (K–P) of DL1 PNs expressing UAS-GFP-αTub84B in WT (E–G, K–M) and TBCD1 (H–J, N–P) at 72 h APF (E, H, K, N), within 1 d after eclosion (F, I, L, O) and 7–10 d adult (G, J, M, P). PN morphologies are labeled in red, green shows GFP-αTub84B staining, and blue shows PNA-biotin staining. Q, R, UAS-HA-syt was expressed in WT (Q) and TBCD1 (R) DL1 PNs. Q, HA-syt normally accumulates in presynaptic sites, the calyx of the MB and the LH. R, HA-syt mislocalizes at the stalk of axon (arrowheads). MB and LH are marked with yellow and white-dotted circles, respectively in A, B, and K–R. Asterisks, dotted circles, and arrows in C–J denote cell bodies, the DL1 glomerulus, and ectopic dendrite arborization, respectively. Scar bars, 20 μm.

Article Snippet: We used rat anti-mCD8 (1:200; Invitrogen, MCD0800), mouse anti-Bruchpilot [1:40; Developmental Studies Hybridoma Bank (DSHB), nc82], rat anti- D N-cadherin (1:40; DSHB, DNEX-8), mouse anti-myc (1:1000; Invitrogen, 46-0603), rabbit anti-GFP (1:500; MBL, 598), mouse anti-HA (1:1000; Covance, 16B12), PNA-biotin (1:250, J-Oil Mills, J214) and mouse anti-FasII (1:40, DSHB, 1D4).

Techniques: Labeling, Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 controls the germinal center by influencing the numbers and activity of T follicular helper cells

doi: 10.4049/jimmunol.1302418

Figure Lengend Snippet: WT and KO mice were immunized with NP-KLH in CFA s.c. 7 days later dLN were isolated and analyzed. A) frozen lymph node sections, 7 days post immunization were stained with PNA (green) and CD4 (red) to visualize germinal centers using confocal microscopy (20x). Imaris software was used to calculate the area of each germinal center, the graph shows GCs measured from 3 independent experiments, each data point is 1 GC B) representative flow cytometry dot plots showing TFH (CD4+ CD44hi CXCR5+ PD-1+) percentage in the dLN 7 days post immunization. C) absolute number of TFH cells in the dLN over the timecourse of the immunization. D) shows GC B cell numbers (CD19+ FAS+ GL-7+) in the dLN of WT and KO mice 11 days post immunization. E) serum anti-NP IgG antibody levels 14 days post immunization were measured by ELISA. *P<0.05; **P<0.01 (graphs show mean ± S.E.M and each data point represents 1 mouse in panels C, D and E)

Article Snippet: Antibodies used to stain for markers are as follows; anti-mouse CD4 (Ebioscience), PNA-biotin (Biosearch technologies), anti-rat AF647, streptavidin AF555 (both Life Technologies, Carlsbad, CA).

Techniques: Isolation, Staining, Confocal Microscopy, Software, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Role of the Transcriptional Corepressor Bcor in Embryonic Stem Cell Differentiation and Early Embryonic Development

doi: 10.1371/journal.pone.0002814

Figure Lengend Snippet: (A) The Bcor Neo/Y and Bcor Gt/Y 129 strain derived ES cells can contribute to the B and T lineages. White blood cells from the spleens of Bcor Neo/Y and Bcor Gt/Y and appropriate controls were stained with antibodies to the 129 strain specific surface antigen Ly9.1 and the T cell markers CD4 and CD8 and B cell marker CD19. Cells were analyzed by flow cytometry and results are displayed as density plots with black boxes gating T or B cell populations of 129 origin. B6 and 129 strain wild type mice control for Ly9.1 specificity. CJ7 and E14 129 mouse strain derived ES cells were used to generate control chimera 1 (Con. Ch. 1) and control chimera 2 (Con. Ch. 2). Flow cytometry analyses were performed on chimeric control mice and on three mice for each mutant chimeric line, of which an example scatter plot is displayed for each. (B) Compared to chimeric controls, Bcor Neo/Y and Bcor Gt/Y 129 strain derived ES cell contribution to B and T cells is reduced. Graphs of scatter plot data show 129 strain contributions to each hematopoietic lineage. Black bars indicate the average contribution of control chimeric mice 1 and 2 (Con, black +) and the combined average of three Bcor Neo (Neo, red +) and three Bcor Gt (Gt, blue +) mice. Contribution to germinal center B cells is also reduced compared to controls as indicated by the final plot showing the PNA+ (peanut agglutinin; germinal center marker) percentage of CD19+ 129 derived B cells. P values were ≤0.05 for all hematopoietic cell populations tested (CD4; 0.004, CD8; 0.034, CD19; 0.023 and PNA; 0.001). (C) Bcor Neo/Y and Bcor Gt/Y ES cells can contribute to adult erythrocytes in chimeric mice but contribution is reduced in comparison to controls. Coomassie stained gel of hemolysates from erythrocytes of mice described above in panels A and B. Bcor Neo and Bcor Gt chimeric mice (Neo 1, 2 and 3 and Gt 1, 2 and 3) primarily express B6 derived ß -single globin where as chimeric control mice 1 and 2 primarily express 129 derived ß -major and ß -minor globin. The hemoglobin control lane (Hbb Con) was prepared from samples that contain ß -single, ß -major and ß -minor globin.

Article Snippet: Cells were stained with a 1∶200 dilution of appropriate primary antibody or lectin (Anti-Ly9.1-FITC (BD Pharmingen), Anti-CD19-PE (eBioscience), Anti-CD4-PerCP (BD Pharmingen), Anti-CD8-APC (eBioscience) and PNA-biotin (BD Pharmingen)) in FACs buffer for 20 minutes at room temperature and washed 1 time with 200 uL of FACs buffer.

Techniques: Derivative Assay, Staining, Marker, Flow Cytometry, Mutagenesis