Journal: PLoS ONE

Article Title: Classical Photoreceptors Are Primarily Responsible for the Pupillary Light Reflex in Mouse

doi: 10.1371/journal.pone.0157226

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Melanopsin , a synthetic peptide consisting of 1–15 N-terminal amino acids (MDSPSGPRVLSSLTQ)of mouse melanopsin , Advanced Targeting Systems; #UF006 , Rabbit, polyclonal , 1:7500 (immunohistochemistry).

Techniques: Immunohistochemistry, Western Blot, Recombinant, Derivative Assay

Journal: PLoS ONE

Article Title: Classical Photoreceptors Are Primarily Responsible for the Pupillary Light Reflex in Mouse

doi: 10.1371/journal.pone.0157226

Figure Lengend Snippet: Three of the 6 mice shown in , which had both MNU and NaIO 3 injected, were administered a melanopsin antagonist (AA41612). This resulted in further reduction in PLR at high irradiances. A two-way ANOVA with repeated measures showed a significant difference between the three groups (F 2,24 = 31.3; P = 0.004). A post hoc Holm-Sidak test showed that injecting AA41612 resulted in significantly lower PLR at irradiances >10 3 μW/cm 2 compared with mice injected with MNU and NaIO 3 . PLR data for wild-type mice is replotted from for comparison.* P <0.05.

Article Snippet: Melanopsin , a synthetic peptide consisting of 1–15 N-terminal amino acids (MDSPSGPRVLSSLTQ)of mouse melanopsin , Advanced Targeting Systems; #UF006 , Rabbit, polyclonal , 1:7500 (immunohistochemistry).

Techniques: Injection, Comparison

Journal: PLoS ONE

Article Title: Classical Photoreceptors Are Primarily Responsible for the Pupillary Light Reflex in Mouse

doi: 10.1371/journal.pone.0157226

Figure Lengend Snippet: (A) Representative immunoblots for melanopsin and the corresponding β-tubulin for wild-type, rd1 and MNU-injected mice. Melanopsin immunoblot produced two bands: glycosylated protein at 85 kDa and unglycosylated protein at 53 kDa. (B) Optical density ratio (melanopsin to β-tubulin) for glycosylated and unglycosylated melanopsin (mean±SE). The levels of glycosylated and unglycosylated melanopsin in rd1 (0.54 ± 0.08 and 0.72 ± 0.07, respectively) and MNU-injected mice (0.84 ± 0.15 and 0.8 ± 0.11) were statistically similar to those in wild-type (0.82 ± 0.13 and 0.86 ± 0.16) (F 2,9 = 1.8, P = 0.22 and F 2,9 = 0.36, P = 0.7, respectively; one-way ANOVA; n = 4 mice). (C) Relative levels of melanopsin mRNA in rd1 (0.83 ± 0.09) and MNU-injected mice (0.92 ± 0.1) were statistically similar to those in wild-type (1.01 ± 0.04) (F 2,30 = 1.25, P = 0.3; one-way ANOVA; n = 11 mice).

Article Snippet: Melanopsin , a synthetic peptide consisting of 1–15 N-terminal amino acids (MDSPSGPRVLSSLTQ)of mouse melanopsin , Advanced Targeting Systems; #UF006 , Rabbit, polyclonal , 1:7500 (immunohistochemistry).

Techniques: Western Blot, Injection, Produced

Journal: PLoS ONE

Article Title: Classical Photoreceptors Are Primarily Responsible for the Pupillary Light Reflex in Mouse

doi: 10.1371/journal.pone.0157226

Figure Lengend Snippet: A) Representative images from flatmounted retinas double-labeled for melanopsin ( green ) and Brn3b ( red ), showing non-M1 cells ( arrows ) in wild-type (WT, left ), rd1 ( middle ) and MNU (7 days post injection; right ). Top row shows original images, middle row shows dendritic stratification of the marked non-M1 ( arrow ) cells, and bottom row shows the same images with melanopsin signal digitally removed to more clearly show the Brn3b labeling in the non-M1 cells. The middle images show 2 thin solid lines marking the boundaries of the IPL and a thin dashed line separating the Off (top) and On (bottom) sublaminas in the IPL. The faintly-labeled non-M1 cells ( arrows ) stratified in ON sublamina and expressed Brn3b in wild-type, rd1 and MNU-injected mice. Scale bar: 50 μm. B) Representative images from flatmounted retinas double-labeled for melanopsin ( green ) and Brn3b ( red ), showing M1 cells ( arrows ) in wild-type ( left ), rd1 ( middle ) and MNU (7 days post injection; right ). Top row shows original images, middle row shows dendritic stratification of the marked M1 ( arrow ) cells, and bottom row shows the same images with melanopsin signal digitally removed to more clearly show the Brn3b labeling in the M1 cells. The middle images show 2 thin solid lines marking the boundaries of the IPL and a thin dashed line separating the Off (top) and On (bottom) sublaminas in the IPL. The brightly-labeled M1 cells ( arrows ) stratified in OFF sublamina and expressed Brn3b in rd1, but not in wild-type or MNU-injected mice. Scale bar: 50 μm. C) The density of mRGCs (mean ± SE) in wild-type, rd1 and MNU-injected mice. A one-way ANOVA showed significant differences among the 3 groups (F 2,33 = 8.18; P = 0.001; n = 8 mice for wild-type and rd1, n = 20 for MNU-injected). A post hoc Holm-Sidak test revealed that both rd1 and MNU-injected mice had significantly higher numbers of mRGCs than wild-type. * P <0.05; † P <0.001. D) The numbers of non-mRGCs, identified by presence of Brn3a immunoreactivity, in wild-type, rd1, and MNU-injected mice were statistically similar (F 2,5 = 1.16; P = 0.39; one-way ANOVA; n = 3 mice for wild-type and rd1, n = 2 for MNU-injected). E) Densities of M1 type of mRGCs in wild-type, rd1 and MNU-injected mice were similar (F 2,33 = 0.32, P = 0.73; one-way ANOVA), whereas the densities of non-M1 cells were different (F 2,33 = 9.74, P <0.001). A post hoc Holm-Sidak test showed that both rd1 and MNU-injected mice had significantly higher numbers of non-M1 cells than in wild-type (n = 8 mice for wild-type and rd1, n = 20 for MNU-injected). The MNU-injected group included animals in which mRGCs were counted at 3, 7, 14 or 28 days after the MNU injection (n = 5 mice each); data from all samples were pooled, because there was no statistical difference among the subgroups (data not shown). * P <0.05, † P <0.001. F) There was a significant difference in the proportion of M1 cells that expressed Brn3b among wild-type, rd1 and MNU-injected mice (F 2,32 = 13.39, P <0.001, one-way ANOVA; n = 8 mice for wild-type, 7 for rd1, 20 for MNU). A post hoc Holm-Sidak test showed that the numbers in rd1 mice, but not in MNU-injected mice, were significantly higher than in wild-type. † P <0.001.

Article Snippet: Melanopsin , a synthetic peptide consisting of 1–15 N-terminal amino acids (MDSPSGPRVLSSLTQ)of mouse melanopsin , Advanced Targeting Systems; #UF006 , Rabbit, polyclonal , 1:7500 (immunohistochemistry).

Techniques: Labeling, Injection

Journal: Scientific Reports

Article Title: Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone

doi: 10.1038/srep14096

Figure Lengend Snippet: ( a , b ) An epifluorescence image of RPE-1 cells showing the TZ protein TMEM67 (green) sandwiched between centrin-eGFP (blue) and acetylated tubulin (red). Scale bar: 2 μm. ( c , d ) Comparison of confocal and STED images of TCTN2 and TMEM67 illustrating the ability of STED to resolve two separate intensity peaks. ( e , j ) A set of STED images showing distinct lateral dimensions of TZ and TF proteins. CEP290 and RPGRIP1L had narrow widths, while MKS1, TMEM67, TCTN2, and CEP164 had separable intensity peaks in the order of increasing widths. Scale bar, 200 nm. ( k ) Lateral widths (mean ± standard deviation) of TZ/TF proteins. ( l ) Distributions of lateral diameters of TZ/TF proteins defined by lateral distances between two intensity peaks.

Article Snippet: For the N-terminal CEP290 antibody, a synthetic peptide consisting of residues 11–24 of mouse Cep290 with a C-terminal cysteine added for conjugation to KLH (IKVDPDDLPRQEEL-C) was used to generate rabbit polyclonal antibodies (Genscript).

Techniques: Comparison, Standard Deviation

Journal: Scientific Reports

Article Title: Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone

doi: 10.1038/srep14096

Figure Lengend Snippet: ( a – f ) Sample dual-color STED images showing different axial positions relative to the distal edge of centrin for different TZ/TF proteins. The axial distance of each BD V500-labeled TZ/TF protein to the FWHM-defined boundary of centrin-eGFP was measured. Centrin-eGFP signal could also be seen in the BD V500 channel. ( g – l ) Comparison of the axial positions of TZ/TF proteins (green) relative to centrin (yellow) revealing that TMEM67 and TCTN2 were dwelled at a similar axial level to MKS1 and RPGRIP1L in terms of the distance to centrin (red dashed line), while CEP290 was at another axial level close to centrin, distinct from the other TZ proteins. CEP164 was slightly proximal to the distal edge of centrin. Scale bar for ( a – l ): 200 nm. ( m ) Comparison of the axial distances to centrin. RPGRIP1L, MKS1, TMEM67, and TCTN2 were ~150 nm from the centrin edge, while CEP290 was ~40 nm from the centrin edge. ( n ) A series of single-color STED images of TZ/TF proteins were axially positioned based on the average relative distances to the distal edge of centrin from dual-color STED images. The threshold of each single-color image was adjusted based on the FWHM to represent their shape and size. These axially-positioned images were overlapped to create a 7-color superresolution image of proteins at the ciliary base. To obtain a white-background image to be merged with an EM image, colors of the merged multi-color image were inverted with a corresponding negative mapping (upper). Scale bar: 200 nm.

Article Snippet: For the N-terminal CEP290 antibody, a synthetic peptide consisting of residues 11–24 of mouse Cep290 with a C-terminal cysteine added for conjugation to KLH (IKVDPDDLPRQEEL-C) was used to generate rabbit polyclonal antibodies (Genscript).

Techniques: Labeling, Comparison

Journal: Scientific Reports

Article Title: Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone

doi: 10.1038/srep14096

Figure Lengend Snippet: ( a ) A 7-color superresolution image obtained by merging multiple single-colored STED images illustrating the relative locations of important TZ/TF proteins. ( b ) A typical equally-magnified EM image of a primary cilium in an RPE-1 cell, where TFs are marked as blue dots, areas of microtubule doublets (MT) circled with red dashed lines, areas of the ciliary membrane (CM) covering the ciliary necklace (CN) circled with yellow dashed lines, and the distal end of the BB marked by a white dashed line. CP: ciliary pocket. ( c ) A merged image of ( a , b ) obtained by aligning the TFs in EM and CEP164 in STED. RPGRIP1L was close to the microtubule doublets; TMEM67 and TCTN2 were localized mostly toward the TZ membrane, while MKS1 was localized midway between the membrane and microtubules. CEP290, right above the BB, was localized at a different axial level from the other TZ proteins. Scale bar, 200 nm. ( d ) A localization model of TZ/TF proteins at the ciliary base pinpointing the positions of these proteins relative to each other and to known structural elements. Y-links are hypothesized to bridge across the level where RPGRIP1L and MKS1 are occupied, with dwelling transmembrane proteins such as TMEM67 and TCTN2 decorated as the ciliary necklace.

Article Snippet: For the N-terminal CEP290 antibody, a synthetic peptide consisting of residues 11–24 of mouse Cep290 with a C-terminal cysteine added for conjugation to KLH (IKVDPDDLPRQEEL-C) was used to generate rabbit polyclonal antibodies (Genscript).

Techniques: Membrane