Journal: STAR Protocols
Article Title: Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve
doi: 10.1016/j.xpro.2021.100555
Figure Lengend Snippet: Quantification of mRNA puncta in sciatic nerves using IMARIS (A) Generate 3D virtual puncta for Ppib mRNA using the 'Spot' image analysis module of Imaris and 3D virtual surfaces for neuronal processes and Schwann cells using the 'Surface' module of Imaris. (1) - (3) Generate 3D virtual puncta for Ppib mRNA with Ppib RNAscope channel image (red). [Diameters for creation: XY = 0.25 μm, Z = 0.9 μm; filter: ‘Quality > 2.5’]. (1) Ppib RNAscope channel raw image. (2) Virtual puncta for Ppib mRNA (yellow). (3) Merged images of Ppib RNAscope channel and virtual Ppib puncta. (4) – (6) Generate 3D virtual surfaces for neuronal processes with MAP2 immunohistochemistry channel (green). [Enable smooth = true; surface grain size = 0.45 μm; enable eliminate background = true; diameter of largest sphere = 4 μm; intensity threshold = 0.29–43.22; filter: “Number of voxels > 100” ]. (4) MAP2 immunohistochemistry channel raw image. (5) Virtual surfaces for neuronal processes. (6) Merge of MAP2 immunohistochemistry channel and virtual neuronal processes surfaces. (7) – (9) Generate 3D virtual surfaces for Schwann cells with S100 immunohistochemistry channel (blue). [Enable smooth = true; surface grain size = 0.4 μm; enable eliminate background = true; diameter of largest sphere = 2.5 μm; intensity threshold = 3 – 52.6187; filter: “Number of voxels > 100”]. (7) S100 immunohistochemistry channel raw image. (8) Virtual surfaces for Schwann cells. (9) Merge of S100 immunohistochemistry channel and virtual Schwann cell surfaces. (10) Merged images for all channels: Ppib mRNA/opal 570 (red); MAP2/Cy2 (green); S100/Dylight 405 (blue). (11) Merged images for all virtual puncta and surfaces: Ppib puncta (yellow); neuronal processes (green); Schwann cells (blue). (12) Merged images for all channels and virtual masks: Ppib puncta/Ppib RNAscope (red); neuronal processes/MAP2 (green); Schwann cells/S100 (blue). (B) Ppib mRNA punctum (red) colocalized with neuronal processes (green) in 3D is marked with a yellow arrowhead. Ppib mRNA punctum not colocalized with neuronal processes is marked with a blue arrowhead. (C) Ppib mRNA punctum (red) colocalized with Schwann cells (blue) in 3D is marked with a yellow arrowhead. Ppib mRNA puncta not colocalized with Schwann cells are marked with blue arrowheads. Scale bar: 10 μm.
Article Snippet: See Quantification of mRNA puncta in sciatic nerves using IMARIS (A) Generate 3D virtual puncta for Ppib mRNA using the 'Spot' image analysis module of Imaris and 3D virtual surfaces for neuronal processes and Schwann cells using the 'Surface' module of Imaris. (1) - (3) Generate 3D virtual puncta for Ppib mRNA with Ppib RNAscope channel image (red).
Techniques: RNAscope, Immunohistochemistry
![Quantification of mRNA puncta in sciatic nerves using IMARIS (A) Generate 3D virtual puncta for Ppib mRNA using the <t>'Spot'</t> <t>image</t> <t>analysis</t> <t>module</t> of Imaris and 3D virtual surfaces for neuronal processes and Schwann cells using the 'Surface' module of Imaris. (1) - (3) Generate 3D virtual puncta for Ppib mRNA with Ppib RNAscope channel image (red). [Diameters for creation: XY = 0.25 μm, Z = 0.9 μm; filter: ‘Quality > 2.5’]. (1) Ppib RNAscope channel raw image. (2) Virtual puncta for Ppib mRNA (yellow). (3) Merged images of Ppib RNAscope channel and virtual Ppib puncta. (4) – (6) Generate 3D virtual surfaces for neuronal processes with MAP2 immunohistochemistry channel (green). [Enable smooth = true; surface grain size = 0.45 μm; enable eliminate background = true; diameter of largest sphere = 4 μm; intensity threshold = 0.29–43.22; filter: “Number of voxels > 100” ]. (4) MAP2 immunohistochemistry channel raw image. (5) Virtual surfaces for neuronal processes. (6) Merge of MAP2 immunohistochemistry channel and virtual neuronal processes surfaces. (7) – (9) Generate 3D virtual surfaces for Schwann cells with S100 immunohistochemistry channel (blue). [Enable smooth = true; surface grain size = 0.4 μm; enable eliminate background = true; diameter of largest sphere = 2.5 μm; intensity threshold = 3 – 52.6187; filter: “Number of voxels > 100”]. (7) S100 immunohistochemistry channel raw image. (8) Virtual surfaces for Schwann cells. (9) Merge of S100 immunohistochemistry channel and virtual Schwann cell surfaces. (10) Merged images for all channels: Ppib mRNA/opal 570 (red); MAP2/Cy2 (green); S100/Dylight 405 (blue). (11) Merged images for all virtual puncta and surfaces: Ppib puncta (yellow); neuronal processes (green); Schwann cells (blue). (12) Merged images for all channels and virtual masks: Ppib puncta/Ppib RNAscope (red); neuronal processes/MAP2 (green); Schwann cells/S100 (blue). (B) Ppib mRNA punctum (red) colocalized with neuronal processes (green) in 3D is marked with a yellow arrowhead. Ppib mRNA punctum not colocalized with neuronal processes is marked with a blue arrowhead. (C) Ppib mRNA punctum (red) colocalized with Schwann cells (blue) in 3D is marked with a yellow arrowhead. Ppib mRNA puncta not colocalized with Schwann cells are marked with blue arrowheads. Scale bar: 10 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5307/pmc08185307/pmc08185307__gr9.jpg)