Nov 26, 2025
TDA-MIT: In situ sequencing for mouse skin and mouse lung
- Ke Zhang1,2
- 1Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;
- 2Broad Institute of MIT and Harvard, Boston, MA, USA
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Ke Zhang 2025. TDA-MIT: In situ sequencing for mouse skin and mouse lung. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88ybdl2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 25, 2025
Last Modified: November 26, 2025
Protocol Integer ID: 233528
Keywords: TDA, in situ seqeuencing, profiling gene expression, gene expression in intact tissue section, mouse lung this protocol, mouse lung, mouse skin, tda, iterative fluorescence
Abstract
This protocol describes an in situ sequencing (ISS) workflow for spatially profiling gene expression in intact tissue sections, using padlock probe hybridization, rolling circle amplification, and iterative fluorescence imaging for barcode decoding.
Materials
| Poly-D-lysine solution | Thermofisher Scientific | Cat# A3890401 | |
| 16% Paraformaldehyde, PFA | Electron Microscope Sciences | Cat# 15710-S | |
| 1X PBS, 7.4 | Gibco | Cat# 10010-023 | |
| 10X PBS, 7.4 | Gibco | Cat# 70011-044 | |
| SUPERase In RNase inhibitor | Invitrogen | Cat# AM2696 | |
| 1M Tris pH 7.5 | Thermofisher Scientific | Cat# 15567027 | |
| Tween-20 | Calbiochem | Cat# 655206 | |
| 20X SSC buffer | Sigma-Aldrich | Cat# S6639 | |
| OminiPur Formamide | Calbiochem | Cat# 75-12-7 | |
| 10X Antarctic Phosphatase Buffer | New England Biolabs | Cat# B0289S | |
| Antarctic Phosphatase | New England Biolabs | Cat# M0289S | |
| Ribonucleoside vanadyl complex, RVC | New England Biolabs | Cat# S1402S | |
| 10mg/mL Yeast tRNA | Fisher Scientific | Cat# AM7119 | |
| T4 DNA ligase, 5Weiss U/uL | Thermo Scientific | Cat# EL0011 | |
| BSA, molecular biology grade | New England Biolabs | Cat# B9000S | |
| Phi29 DNA polymerase | Thermo Scientific | Cat# EP0094 | |
| 10 mM dNTP mix | Invitrogen | Cat# 18427088 | |
| 5-(3-aminoallyl)-dUTP | Invitrogen | Cat# AM8439 | |
| Methacrylic acid NHS ester | Sigma-Aldrich | Cat# 730300 | |
| Acrylamide, solution 40% | Bio-Rad | Cat# 161-0140 | |
| Bis-acrylamide solution, 2% | Bio-Rad | Cat# 161-0142 | |
| Ammonium persulfate, APS | Sigma-Aldrich | Cat# A3678 | |
| Tetramethylethylenediamine, TEMED | Sigma-Aldrich | Cat# T9281 | |
| Gel-Slick Solution | Lonza | Cat# 50640 | |
| Proteinase K, RNA grade | Invitrogen | Cat# 25530049 | |
| OminiPur SDS, 20% | Calbiochem | Cat# 7991 | |
| 5M NaCl | Thermofisher Scientific | Cat# AM9759 | |
| Shrimp Alkaline Phosphatase | New England Biolabs | Cat# M0371L | |
| CutSmart buffer | New England Biolabs | Cat# B6004S | |
| Triton-X-100, 10% solution | Sigma-Aldrich | Cat# 93443 | |
| AEBSF | Sigma-Aldrich | Cat# A8456-25MG | |
| DAPI | Sigma-Aldrich | Cat# 10236276001 | |
| 100% Ethanol | VWR Life Science | Cat# 89125-170 |
Troubleshooting
Sample preparation
A quartz-bottom culture dish was coated with a poly-D-lysine solution. OCT-embedded mouse lung and skin tissues were sectioned into 14 μm slices, then fixed with 4% PFA in PBS for 15 minutes, followed by three washes with PBSR (PBS + RNase Inhibitor). Fixed samples submerged with PBSR underwent Raman imaging before being permeabilized with pre-chilled methanol at -20°C for 20 minutes, followed by hybridization.
In situ sequencing library construction
SNAIL probes were designed, and the probe library was constructed according to Wang et al.1. The probes were dissolved and pooled in ultrapure RNase-free water. The probe mixture was heated to 40°C for 15 minutes and then equilibrated to 37°C. Tissue samples were removed from -20°C storage, equilibrated to room temperature, and treated with 10 mM Tris pH 7.5 for 10 minutes. The samples were washed with PBSTR (0.1% Tween-20, 0.1 U/μL SUPERase•In in PBS) and incubated in hybridization buffer (2X SSC, 10% formamide, 1% Tween-20, 20 mM ribonucleoside vanadyl complex, 0.1 mg/mL yeast tRNA, 0.1 U/μL SUPERase•In, SNAIL probes (4 nM per probe for STARmap-ISS, 10nM per probe for STARmap-ISH) at 40°C with gentle shaking for 48 hours. The samples were washed twice with PBSTR for 20 minutes at 37°C and then with 4X SSC in PBSTR for 20 minutes at 37°C, followed by a rinse with PBSTR at room temperature. The SNAIL padlock probes annealed to the samples were ligated by incubating with a T4 DNA ligation mixture (1:10 dilution of T4 DNA ligase, 0.2 mg/mL BSA, 0.5 U/μL SUPERase•In) for 2 hours at room temperature with gentle agitation, followed by two 5-minute washes with PBSTR. The samples were then incubated in the RCA mixture (1:50 dilution of Phi29 DNA polymerase, 250 μM dNTP, 20 μM 5-(3-aminoallyl)-dUTP, 0.2 mg/mL BSA, 0.2 U/μL SUPERase•In) for 2 hours at 30°C with gentle agitation, followed by two 5-minute washes with PBSTR. Subsequently, the samples were treated with 25 mM acrylic acid NHS ester for 2 hours at room temperature with agitation, rinsed with PBST (0.1% Tween-20 in PBS) once, and incubated with monomer buffer (4% acrylamide, 0.2% bis-acrylamide, 2X SSC in H2O) for 15 minutes for polymerization pre-treatment. The buffer was removed, and 30 μL of monomer mixture (0.1% ammonium persulfate, 0.1% tetramethylethylenediamine in monomer buffer) was directly added to the center of each sample, which was immediately covered with a coverslip (coated with Gel-Slick Solution according to the manufacturer’s instructions) and allowed to polymerize at room temperature for 1 hour. The tissue-gel hybrid was washed with PBST twice and cleared by proteinase K digestion mixture (50 mM Tris pH 7.5, 100 mM NaCl, 1% SDS, 0.2 mg/mL proteinase K in H2O) at 37°C overnight. On the following day, the samples were treated with a dephosphorylation mixture (1:100 dilution of shrimp alkaline phosphatase, 0.2 mg/mL BSA, 1:10 dilution of CutSmart buffer in H2O) and rinsed with PBST.
Imaging and sequencing
For in situ hybridization detection, the 19-nt fluorescent oligo complementary to the DNA amplicon was diluted to 100 nM in 1X SSC dissolved in PBST, and samples were incubated at room temperature for 30 minutes, then washed with PBST three times for 5 minutes each before imaging. For in situ sequencing detection, the protocol was followed according to Wang et al.1. Each sequencing cycle started with treatment with stripping buffer (60% formamide, 0.1% Triton-X-100) for 5 minutes and triple-washing with PBST for 5 minutes. The samples were then incubated with a sequencing mixture (0.2 mg/mL BSA, 10 μM reading probe, 5 μM fluorescent decoding probe, 1:25 dilution of T4 DNA ligase) for 3 hours at room temperature. Subsequently, the samples were triple washed with washing and imaging buffers (2X SSC, 10% formamide) for 10 minutes before imaging. DAPI staining was performed after Cycle 6 of imaging for cell segmentation. Images were acquired using a Leica Stellaris5 confocal microscope with a 405 nm diode, white light laser, and a 20x air objective (NA 0.75).
Protocol references
1. Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, (2018).