mouse anti islet 1 40 2d6  (Developmental Studies Hybridoma Bank)

Journal: BMC Developmental Biology

Article Title: Eye and neural defects associated with loss of GDF6

doi: 10.1186/1471-213X-6-43

Figure Lengend Snippet: Loss of retinal differentiation and laminar structure in GDF6 depleted embryos . Histology of stage 41 embryos with small eyes following GDF6 MO injection (20 ng) compared with normal stage matched controls. DAPI staining of small eye (B, D, F, and H) compared with normal eye (A, C, E, and G) showing small eyes have a lack of laminar structure and appear disorganized. (A, B) Photoreceptors (p) are not stained in B compared with XAP-1 staining in A. (B) Cell bodies are visibly extending outward (white arrow) but do not stain for XAP-1. Red staining next to the lens is non-specific staining also detected in a proportion of negative controls. (C, D) 40.2D6 detects ganglion (g) and amacrine (a) cells within the normal retina, but these cells are not detected in the small eye. Photoreceptors are visible due to autofluorescence at increased exposure. (E, F) Small disorganized eyes show a loss of immunostaining for the cytoplasmic domain of NCAM (n). (G, H) Mitotic cells (m) detected using anti-phosphorylated-histoneH3 are few and found mainly in the ciliary marginal zone in normal stage 41 embryos. The small eye shows a few mitotic cells near the RPE layer.

Article Snippet: Ganglion and Amacrine cells, Photoreceptor cells, NCAM and mitotic cells were detected with 1:3 anti-islet 1 (40.2D6 supernatant – DSHB), 1:20 anti-XAP (3D2 supernatant – DSHB), 1:20 anti-NCAM (4D supernatant – DSHB) and 1:100 anti-phospho-Histone H3 (Upstate) respectively.

Techniques: Injection, Staining, Immunostaining

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Retinal differentiation from ISL1 −/− hESC (A) Bright field image of WT and ISL1 −/− hESC-retinas showing the aggregation on 96 well V-plate (DD15). (B) Immunostaining of the DD15 WT and ISL1 −/− hESC-retina for expressed Chx10. (C) At DD88, self-organized retinas with a continuous epithelial structure were consistently differentiated from WT and ISL1 −/− hESC with Crx:: Venus expression. (D) WT and ISL1 −/− hESC-retinas were stained with ISL1 and Brn3 (DD60). (E) FCM analysis of ISL1 and Crx population on WT and ISL1 −/− hESC-retina (DD54). (F and G) Temporal population changes in Brn3 + RGCs and Crx:: Venus + photoreceptors of WT (blue) and ISL1 −/− (orange) hESC-retinas quantified by FCM (n = 3 for each). (H) Population of RPC (Crx − /Chx10 + ), RGC (Brn3 + ), RGC and AC (Pax6 ++ ), and cone photoreceptors (Crx + /Rxrγ + ) in WT and ISL1 −/− hESC-retina quantified by FCM at DD58 (n = 4 for each). (I) Crx:: Venus + hESC-retinas with cilia-like structures on the surface in long-term culture at DD235 (arrows). (J–P) Representative immunostaining and FACS plot for WT and ISL1 −/− hESC-retinas at around DD240. (J–L) Photoreceptors formed ONL-like structure with a similar differentiation rate of photoreceptors. (M–P) ON/rod-bipolar cell populations with PKCα, L7, and Goα expression were diminished in ISL1 −/− hESC-retina (n = 3 for each). Data are presented as means ± SEM.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Immunostaining, Expressing, Staining

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Maturation of hESC-retina after transplantation in RD-nude rats (A) Schematic illustration of subretinal transplantation of hESC-retina. (B) In vivo fundus imaging of transplanted Crx:: Venus + hESC-retina on 2, 4, and 24 weeks after transplantation. (C and D) Immunostaining for rat retina with S-opsin, L/M-opsin, and rhodopsin in WT and ISL1 −/− hESC-retinas after 24 weeks of transplantation with DAPI nuclear staining. (E and F) Ku80 + /ISL1 + human inner cells (arrows) surround graft photoreceptors in the WT but were absent in ISL1 −/− hESC-retina. (G–J) Goα + or PKCα + ON/rod-bipolar cells were present in the Ku80 + WT but not in ISL1 −/− hESC-retina. (K and L) Note that host bipolar dendrites were observed surrounding the graft photoreceptors (yellow arrows). A few Chx10 + /PKCα − cells were present in HuNu positive graft cells in ISL1 −/− hESC-retina (white arrows). DD, differentiation day; TP, transplantation; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Transplantation Assay, In Vivo, Imaging, Immunostaining, Staining

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Expression of phototransduction protein and IPM protein in ISL1 −/− hESC-derived photoreceptors after transplantation (A) Summary of phototransduction signaling cascades. (B) Expression of rod phototransduction proteins (a–g), cone phototransduction proteins (h–m), and guanylyl cyclase (GC) and GC-activating proteins (n–q) in the Crx:: Venus + photoreceptor rosettes. Maturation of photoreceptors with IS/OS formation was indicated by PRPH2 (r). Expression of IPM proteins was found in the rosette (s–x). (C and D) Expression of IRBP, IMPG1(C), and GNAT1 (D) in the ISL1 −/− hESC graft.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Expressing, Derivative Assay, Transplantation Assay

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Histological pattern comparison between the WT and ISL1 −/− retina grafts (A, E, and I) Schematic representations of three patterns: poor , fair , and good for rosette contact. (A) IPL preservation (E) and OS elongation (I). (B, F, and J) Summary of data (bars) indicating the rate of poor , fair , and good per sample (transplanted retina), along with model predictions (dot and bars indicating mode and 95% compatibility interval) for rosette contact (B), IPL preservation (F), and OS elongation (J). (C, G, and K) Distributions of predicted mean (top: predicted mean) and expected distribution (bottom: predicted distribution) for rosette contact. (C) IPL preservation (G) and OS elongation (K). (D, H, and L) Posterior distribution of model parameters: rosette contact. (D) IPL preservation (H) and OS elongation (L). Bars above graphs indicate mode and 95% compatibility interval. A total of 541 rosettes (311 WT and 230 ISL1 −/− rosettes, from 10 WT to 12 ISL1 −/− transplanted retinas) were used for rosette contact analysis, 493 IPL areas under rosettes (222 WT and 271 ISL1 −/− areas under rosettes, from 10 WT and 13 ISL1 −/− hESC-retinas) were used for the IPL analysis, and 614 rosettes (298 WT and 316 ISL1 −/− rosettes, from 10 WT and 13 ISL1 −/− hESC-retinas) were used for the OS analysis. BP, bipolar cells; PR, photoreceptors; AC, Amacrine cells.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Preserving

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Host-graft synaptic formation of transplanted photoreceptors derived from ISL1 −/− hESC-retina (A and B) Presynaptic marker CtBP2 was localized on the margin of the Crx:: Venus + /Recoverin + photoreceptors and the dendritic tips of Ku80 - /PKCα + host bipolar cells (yellow arrows). (C) Outer Plexiform layer-like structures formed with synaptophysin (Syn) around the rosette. (D–F) Presynaptic proteins pikachurin, CtBP2, PSD95, and LRIT3 were present at the dendritic tips of PKCα + host rod bipolar cells. (G) Postsynaptic marker CACNA1S were coupled with pikachurin. (H) Schematic illustration showing the major elements in the photoreceptor-ON-bipolar synapses. (I) Putative CtBP2/mGluR6 synaptic complexes were present at the extended dendritic tips of PKCα + host rod bipolar cells. (J) Putative PNA/mGluR6 cone photoreceptor synapse formation was observed at the dendrite tips of Secretagogin + cone bipolar cells. (K–L) Host (Ku80 − ) and graft (Ku80 + ) Calbindin + horizontal cells dendrites were observed surrounding graft rosette.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Derivative Assay, Marker

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Light responses of transplanted retina by MEA recordings (A–D) Representative recordings of transplanted ( ISL1 −/− ) hESC-retina using MEA system. Transplanted rat retina (A) was mounted on the MEA probe with the Crx:: Venus + hESC-retina centered on electrode area (B) indicated by red boxes. (C) Peri-stimulus time histogram of host RGC spikes with responses to strong light stimuli after washout of L-AP4. Detailed raster plots from three channels (highlighted in black, red, and green) are shown in (D). (E) Population averages of transplanted retinas with WT and ISL1 −/− hESC-retinas and nontransplanted control retinas. Thin lines represent the sample average and thicker lines represent the group averages for the respective L-AP4 treatment condition (before, L-AP4, after) and light stimulation (weak, medium, strong, super-strong). (F) Breakdown of the functional RGC types detected in both transplanted and control retinas. (G) Distribution of RGC spontaneous firing (log(Hz)). Vertical lines indicate the estimated overall mean (1.68). Bars summarize recorded data, whereas lines and ribbon plots show the mode and 95% compatibility interval of model posterior predictions. (H) Posterior distribution of model parameters for RGC spontaneous activity. (I) Summary of RGC response probability with light stimulation. Dots and bars show per sample summary of collected data with lines showing the Clopper and Pearson binomial 95% confidence interval. Violin plots show the model posterior predictions. (J) Posterior distribution of model parameters for RGC responsiveness. Note that values represent log odds. These data in this figure was collected from a total of 47 retinas (16 control, 13 WT, and 18 ISL1 −/− graft transplanted retinas).

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Functional Assay, Activity Assay

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet: Factors affecting light responsiveness after transplantation (A) Pair plot of light responsiveness, spontaneous activity, rosette contact, IPL preservation, and OS elongation. Per sample predictions from the respective analyses were used to analyze correlation between these five features: responsive (estimated light response probability); spontaneous (estimated mean (log) spontaneous firing frequency); contact, OS, and IPL (the estimated mean of the underlying metric variable for the ordinal probit regression, with higher values indicating better performance). In addition to the features mentioned earlier, sex (female or male) and group (control, WT, and ISL1 −/− ) breakdowns are also shown. The diagonal shows the distribution of values (histograms for continuous variables and bar chart for categorical variables). The plots below the diagonal show scatterplots, and the upper triangular plots show the posterior estimates of correlation coefficients with mode and 95% interval indicated on top or the per category breakdown of the distribution for categorical variables. Control samples are shown in gray, WT in blue, and ISL1 −/− in orange. (B) Schematic illustration of a summary of our results. ISL1 −/− hESC-retina (right) has reduced graft bipolar cells, a greater number of host-graft cell contact, and better RGC light responses compared with WT hESC-retina (left) after transplantation.

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Transplantation Assay, Activity Assay, Preserving

Journal: iScience

Article Title: A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation

doi: 10.1016/j.isci.2021.103657

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Islet-1 , Developmental Studies Hybridoma Bank (DSHB) , Cat#40.2D6 RRID: AB_528315.

Techniques: Recombinant, Knock-Out, Software, Imaging

Journal: The Journal of Comparative Neurology

Article Title: Axon guidance at the spinal cord midline—A live imaging perspective

doi: 10.1002/cne.25107

Figure Lengend Snippet: Patterning and morphology of cultured intact spinal cords were conserved after 1 day ex vivo. After intact HH22 spinal cords were cultured and imaged for 1 day ex vivo, they were fixed and transverse cryosections were immunostained for different dorsal and ventral patterning markers, and counterstained with Hoechst. (a) The dI1 interneuron marker Lhx2 confirmed that these neurons were still localized in the most dorsal part of the spinal cord, as expected (black arrowheads). (b) Islet‐1 was used as a marker for dorsal root ganglia (DRG) neurons (magenta asterisks), dI3 interneurons (black arrows) and motoneurons (black arrowheads). All of them maintained the appropriate position: clustered DRG neurons adjacent to the spinal cord; dI3 interneurons localized ventrally of dI1 interneurons; motoneurons on both sides of the ventral spinal cord. (c) Nkx2.2 staining was used to reveal the ventral population of V3 progenitors that are just next to the FP and form the typical inverted V‐shape (black arrow). (d) Finally, FP cells forming the intermediate target for dI1 axons were visualized with Hnf3β staining. They were localized at the ventral midline of the spinal cord as expected (black arrow). (e) Intact spinal cord cultured for 24 h maintained morphology and localization of cell types as illustrated by staining for BEN: motor neurons (black arrowheads) and their ventral roots (magenta asterisks, the dorsal roots (black asterisks), and the dorsal funiculi (black arrows) formed by DRG afferents. (f) BEN staining also revealed that the FP maintained its triangular shape (dashed lines) and its bulky basal segment (white arrow). (g) Same staining of open‐book preparations, which were cultured for the same amount of time: the motor column (black arrowhead) is stained for BEN but ventral roots and DRG afferents were removed during dissection. (h) The FP (dashed line) lost its triangular shape and its basal segment appeared to be much narrower in cultured open‐books (white arrow). (i) Overview image of an intact spinal cord cultured for 24 h and stained for laminin, revealing intact meninges surrounding the spinal cord (black arrowheads). (j) Higher magnification image showing intact laminin‐positive basal lamina (white arrowheads) at the ventral midline. (k) Overview image of an open‐book cultured for 24 h showing the lack of meninges on the basal side of the spinal cord (black arrowheads) and a patch of meninges that remained below the FP (black arrow). (l) Higher magnification of the same section revealed that the laminin‐enriched basal lamina was intact but deformed at the level of the FP (white arrow) and discontinued around it (white arrowheads). (m,n) Staining for cleaved caspase revealed a weak signal in the DRG (black asterisks) and a very weak signal in the motor column (arrowheads) of a cultured intact spinal cord. (o,p) The same staining showed a very strong signal in the ventral spinal cord of a cultured open‐book (arrowheads). Hoechst was used to counterstain nuclei. The dashed black lines represent the outer border of the spinal cord. Note that small images on the upper right corner of the different panels show the β‐actin::EGFP‐F‐electroporated side of the spinal cord. When not indicated dorsal is up. DEV, day ex vivo; d, dorsal; v, ventral, Cleav., Cleaved. Scale bars: 50 μm (a–e,g,I,k,m,o) and 25 μm (f,h,j,l,n,p)

Article Snippet: Primary antibodies were diluted in blocking buffer and added to sections overnight at 4°C (1:400 for goat‐anti‐GFP‐FITC, Cat# 600‐102‐215, RRID: AB_218187, Rockland; 1:2500 for rabbit‐anti‐RFP, Cat# ABIN129578, RRID: AB_10781500, antibodies‐online; 1:200 for rabbit‐anti‐cleaved caspase 3, Cat# 9661S, RRID: AB_2341188, Cell Signaling; 1:500 for goat‐anti‐NrCAM, G68, polyclonal antibody produced against the full length of chicken NrCAM purified from E14 chicken brain membranes (Fitzli et al., ); supernatants containing monoclonal mouse antibodies obtained from DSHB: anti‐Lhx2 (clone PCRP‐LHX2‐1C11, RRID: AB_2618817), anti‐islet‐1 (clone 40.2D6, RRID: AB_528315), anti‐Nkx2.2 (clone 74.5A5, RRID: AB_531794), anti‐Hnf3β (clone 4C7, RRID: AB_2278498); 3.1 μg/ml of mouse‐anti‐Shh (clone 5E1, RRID: AB_2188307); mouse‐anti‐laminin‐1 (clone 3H11, RRID: AB_528342); mouse‐anti‐BEN/SC‐1 (clone BEN, RRID: AB_2314001)).

Techniques: Cell Culture, Ex Vivo, Marker, Staining, Dissection