Multiplex Fluorescent In Situ Hybridization

Samples were fixed in either 10% neutral buffered formalin, dehydrated with ethanol and embedded in paraffin wax or fixed in 4% paraformaldehyde and embedded in OCT compound. Sections from paraffin (5 μm) and OCT (20 μm) blocks were processed using standard pre-treatment conditions for each per the RNAscope multiplex fluorescent reagent kit version 2 (Advanced Cell Diagnostics) assay protocol. TSA-plus fluorescein, Cy3 and Cy5 fluorophores were used at 1:500 dilution. Micrographs were acquired with a laser scanning confocal fluorescence microscope (Zeiss LSM780) and processed with ImageJ and Imaris (version 9.2.0, Oxford Instruments). smFISH experiments were performed on at least 2 human or mouse subjects distinct from the donors used for sequencing, and quantifications were based on at least 10 fields of view in each. For smFISH, fields of view were scored manually, calling a cell positive for each gene probed if its nucleus had >3 associated expression puncta. Proprietary (Advanced Cell Diagnostics) probes used were: KRT5 (547901-C2), SERPINB3 (828601-C3), SFTPC (452561-C2), WIF1 (429391), CLDN5 (517141-C2, 517141-C3), MYC (311761-C3), ACKR1 (525131, 525131-C2), COL1A2 (432721), GPC3 (418091-C2), SERPINF1 (564391-C3), C20rf85 (560841-C3), DHRS9 (467261), GJA5 (471431), CCL21 (474371-C2), COX4I2 (570351-C3), APOE (433091-C2), ACGT2 (828611-C2), ASPN (404481), IGSF21 (572181-C3), GPR34 (521021), EREG (313081), GPR183 (458801-C2), TREM2 (420491-C3), CHI3L1 (408121), MYRF (499261), AGER (470121-C3), TBX5 (564041), KCNK3 (536851), ACVRL1 (559221), SERPINA1 (435441), HHIP (464811), Slc7a10 (497081-C2), Fgfr4 (443511), Pi16 (451311-C2), Serpinf1 (310731), Hhip (448441-C3), Sftpc (314101-C2), Nkx2–1 (434721-C3), and Myrf (524061).

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Travaglini K.J., Nabhan A.N., Penland L., Sinha R., Gillich A., Sit R.V., Chang S., Conley S.D., Mori Y., Seita J., Berry G.J., Shrager J.B., Metzger R.J., Kuo C.S., Neff N., Weissman I.L., Quake S.R, & Krasnow M.A. (2020). A molecular cell atlas of the human lung from single cell RNA sequencing. Nature, 587(7835), 619-625.

Publication 2020

Ackr1 Acvrl1 Ager Apoe Assay Ccl21 Cell Col1a2 Confocal microscope Dehydrated ethanol Diagnostics Dilution Donors Ereg Fgfr4 Fluorescein Fluorescence Formalin Gene Gpc3 Hhip Human Krt5 Mouse Nkx2 1 Nucleus Paraffin Paraformaldehyde Serpina1 Serpinb3 Trem2

Corresponding Organization :

Other organizations : Howard Hughes Medical Institute, Stanford University, Chan Zuckerberg Initiative (United States)

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Protocol cited in 9 other protocols

Variable analysis

independent variables

Fixation with 10% neutral buffered formalin and embedding in paraffin wax
Fixation with 4% paraformaldehyde and embedding in OCT compound

dependent variables

Expression of target genes (KRT5, SERPINB3, SFTPC, WIF1, CLDN5, MYC, ACKR1, COL1A2, GPC3, SERPINF1, C20rf85, DHRS9, GJA5, CCL21, COX4I2, APOE, ACGT2, ASPN, IGSF21, GPR34, EREG, GPR183, TREM2, CHI3L1, MYRF, AGER, TBX5, KCNK3, ACVRL1, SERPINA1, HHIP, Slc7a10, Fgfr4, Pi16, Serpinf1, Hhip, Sftpc, Nkx2-1, Myrf)
Quantification of cells positive for each gene probe

control variables

Tissue processing and staining protocols (pre-treatment conditions for RNAscope assay)
Image acquisition and analysis (confocal microscopy, ImageJ, and Imaris)

controls

Positive control: not specified
Negative control: not specified

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