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CNE_22429_sm_suppinfoFigure1.tif5266KFigure 1. Diacylglycerol lipase α (DGLα) is present in the postsynaptic terminals of Type 1 OFF cone bipolar cells. (Magenta-green version for the assistance of color blind authors.) A, B) Paired images taken under same settings in mouse retina show DGLα staining alone (A) and DGLα coincubated with immunizing protein (B, IP 5 μg/mL) which blocks the DGLα staining. Scale bar 50 μm. C) Combined fluorescence and Nomarski image shows relative localization of DGLα staining, prominent in clusters in the outer plexiform layer (OPL) and more diffusely distributed in the inner plexiform layer (IPL). Scale bar: 50 μm. D) In a compressed Z-series DGLα (red) does not costain with PKC (green), a marker for rod bipolar cells, in the OPL. Scale bar: 25 μm. E) In higher magnification image of single plane, DGLα (red) clearly does not overlap with PKC (green) in OPL. Scale bar: 15μm. F) DGLα (red) does not colocalize with Gγ13 (green, a marker for ON-bipolar cells). Scale bar: 25 μm. G) DGLα (red) does not overlap with Type 2 OFF bipolar cell marker recoverin (green). Scale bar: 25 μm. H) DGLα (red) colocalizes with post-synaptic terminals of Type 1 OFF bipolar cells, as marked by NK3R (green, arrows). Scale bar: 25 μm. I) CB1 (red) does not colocalize with horizontal cell marker calbindin (green) in OPL. Scale bar: 15 μm. J) Flattened Z-series indicates CB1 (red) closely juxtaposes distal to DGLα (green), consistent with rod spherule and cone pedicle localization. K) CB1 (red) costaining with PSD95, which outlines rod spherules (arrows) and cone pedicles (angled arrows) shows CB1 present within terminals of both rod and cone photoreceptor terminals. Scale bar: 25 μm.
CNE_22429_sm_suppinfoFigure2.tif3039KFigure 2. DGLα labeling is widely distributed throughout the IPL. (Magenta-green version for the assistance of color blind authors.) Aside from the pronounced cone bipolar cell staining, dimmer punctate DGLα is also present in the OPL. A) punctate DGLα is present proximal to rod spherules. In the IPL, DGLα is chiefly present in the distal two-thirds of the IPL, but without prominent lamination. Scale bar: 10 μm. B) DGLα (red) does not costain with putative GAD67-GFP neurons and processes (green). Scale bar: 25 μm. C) DGLα (red) does not costain with CB1 (green). Scale bar: 20 μm.
CNE_22429_sm_suppinfoFigure3.tif3036KFigure 3. MGL is present in rod spherules and cone pedicles in the OPL and is expressed prominently in two laminae of the IPL. (Magenta-green version for the assistance of color blind authors.) A, B) Paired images taken under same conditions in mouse retina show MGL staining alone (A) and MGL coincubated with immunizing protein (B, IP 5 μg/mL) which blocks the staining. Scale bars: 60 μm. C) Combined fluorescence and Nomarski image shows relative localization of MGL staining, most prominent in the two laminae of the IPL (arrows), and in the ganglion cell layer (GCL), but also in the OPL. Scale bar: 50 μm. D) MGL (red) staining with DGLα (green) shows their apparent non-overlap and relative localization. Scale bar: 25μm. E) MGL costaining with GAD6, a marker for inhibitory neurons, shows little if any colocalization. Scale bar: 10 μm. F) MGL (red) costaining with PSD95 (green), a marker that outlines rod and cone terminals shows MGL within a cone terminal (arrow) as well as punctate staining within multiple rod spherules. Scale bar: 15 μm. G) MGL (red) costaining with SV2 (green), a marker for rod spherules, shows substantial overlap in the OPL (arrows). Scale bar: 25 μm.
CNE_22429_sm_suppinfoFigure4.tif3597KFigure 4. ABHD6 staining is widely distributed in IPL, GCL and INL. (Magenta-green version for the assistance of color blind authors.) A) Paired images taken under same settings in mouse retina show ABHD6 staining alone (A) and ABHD6 coincubated with immunizing protein (B, IP 5 μg/mL) which blocks the ABHD6 staining. Scale bars: 50 μm. C) Flattened Z-series shows relative localization of ABHD6 staining, with clearly labeled neuronal populations in the proximal INL (downward arrows) and in the GCL (upward arrowheads). Scale bar: 50 μm. D) Costaining of ABHD6 (green) with DGLα (red) reveals neither colocalization nor apparent juxtaposition. Scale bar: 15 μm.
CNE_22429_sm_suppinfoFigure5.tif5387KFigure 5. ABHD6 is present in calbindin and GAD67-positive amacrine cells as well as dendritic MAP2-positive processes. (Magenta-green version for the assistance of color blind authors.) A) ABHD6 costaining with rod bipolar cell marker PKC shows no overlap with rod bipolar cell terminals or processes. Scale bar: 25 μm. B) Costaining with calbindin reveals clear overlap with calbindin-positive amacrine cells (arrows). Scale bar: 15 μm. C) ABHD6 colocalizes with MAP2 in some processes of the proximal IPL. Scale bar: 10 μm. D) ABHD6 colocalizes with GAD67-GFP presumptive inhibitory amacrine cells in the INL. Scale bar: 40 μm.
CNE_22429_sm_suppinfoFigure6.tif7158KFigure 6. Cannabinoid receptor interacting protein 1a (CRIP1a) expression is limited to a population of calbindin-positive amacrine cells. (Magenta-green version for the assistance of color blind authors.) A, B) Coincubation with immunizing protein (IP 5 μg/mL) blocks the staining (control image taken at same settings). Scale bars: 50 μm. C-F) CRIP1a antibody recognizes HA-hCRIP1a transiently expressed in HEK cells. HEK cells were transiently transfected with HA-tagged hCRIP1a and stained using HA- and hCRIP1a- antibodies. C) DAPI staining identifies cell nuclei (arrowheads). D) HA antibody (red) staining shows two HA-hCRIP1a transfected cells (nuclei indicated by arrows). E) hCRIP1a staining (green) labels the same cells identified with the HA antibody. F) Overlay of HA- and hCRIP1a-images shows both antibodies overlap completely (yellow), DAPI fluorescence identifies all cells in the field. The non-transfected cells are not labeled with the hCRIP1a antibody. Scale bar: 15 μm. G) CRIP1a (red) and calbindin (green) show occasional CRIP1a/ calbindin-positive amacrine cells (arrows). Scale bar: 25 μm. H) The same combination in the OPL shows the relative position of CRIP1a in OPL. Scale bar: 25 μm.
CNE_22429_sm_suppinfoFigure7.tif5799KFigure 7. Cannabinoid receptor interacting protein 1a (CRIP1a) expression is limited to presynaptic terminals of cone photoreceptors juxtaposed to Type 1 OFF cone bipolar cells. (Magenta-green version for the assistance of color blind authors.) A) In a compressed Z-series, CRIP1a (red) juxtaposes distal to DGLα (green) in OPL. Scale bar: 25 μm. B-D) CRIP1a costaining with PSD95, shows that CRIP1a is limited to presynaptic terminal of cone photoreceptors. Scale bar: 5 μm. E) CRIP1a (green) staining is close to that of CB1 (red) in OPL suggesting that both are present in the same cone photoreceptor presynaptic terminals. Scale bar: 5 μm.
CNE_22429_sm_suppinfoFigure8.tif4564KFigure 8. DGLβ is expressed exclusively in blood vessels. (Magenta-green version for the assistance of color blind authors.) A) Image shows DGLβ staining in mouse retina. B) Coincubation with immunizing protein (IP 5 μg/mL) blocks the DGLβ staining (control image taken at same settings). C) Combined fluorescence and Nomarski image shows relative localization of DGLβ staining (arrows). D) Flattened Z-series shows that the staining for DGLβ is restricted to blood vessels. Scale bars: 60 μm.
CNE_22429_sm_suppinfoFigure9.tif4184KFigure 9. Fatty acid amide hydrolase (FAAH) is widely expressed in the inner segments (IS), outer nuclear layer (ONL), and ganglion cell layer (GCL), and in the axon terminals of photoreceptors in the outer plexiform layer (OPL). (Magenta-green version for the assistance of color blind authors.) FAAH is widely expressed in wild-type (WT) mouse retina (A), but not in FAAH knockout (KO) mouse retina (B). Scale bar: 25 μm. C) FAAH (green) costaining with PSD95 (red), a marker that outlines rod and cone terminals, shows FAAH within rod terminals (arrows). Scale bar: 10 μm. D, E) In a compressed Z-series FAAH staining (green, arrows) does not overlap with PKC (red), a marker for rod bipolar cells, in the OPL. Scale bar: 5 μm. F) FAAH (green) is not expressed in calbindin (red)-positive horizontal cell body. G) FAAH (green) also does not costain with calbindin (red)-positive horizontal axonal terminals (arrows). Scale bar: 15 μm. H) Costaining of FAAH (green) and CB1 (red) shows overlap (in yellow) in subpopulation of amacrine cells in INL and in cells in the GCL. Scale bar: 25 μm. I) In a higher magnification image of a compressed Z-series FAAH (green) costains with CB1 (red) (arrows) within rod photoreceptor terminals in OPL (arrows). Scale bar: 10 μm.
CNE_22429_sm_suppinfoFigure10.tif1033KFigure 10. N-acylethanolamine-hydrolyzing acid amidase (NAAA) expression is limited to retinal pigment epithelium (RPE). (Magenta-green version for the assistance of color blind authors.) A-D) NAAA antibody recognizes rNAAA transiently expressed in HEK cells. HEK cells were transiently transfected with FLAG-tagged rNAAA and stained using FLAG and NAAA antibodies. A) DAPI staining identifies cell nuclei (arrowheads). B) FLAG antibody (red) staining shows four FLAG-rNAAA transfected cells (nuclei indicated by arrows). C) rNAAA staining (green) labels the same cells identified with the FLAG antibody. D) Overlay of FLAG- and rNAAA-images shows both antibodies overlap completely, DAPI fluorescence identifies all cells in the field. The non-transfected cells are not labeled with the rNAAA antibody. Scale bar: 15 μm. E) Fluorescence image shows NAAA expression limited to RPE. F) Image taken at same settings of NAAA coincubated with immunizing protein (IP 10 μg/mL). Scale bar: 60 μm.

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