probe set in TE buffer to a final concentration of 25 μM and
store in aliquots protected from light at À20
C.
22. CLN2-Q570 probe set: smFISH probes mixed in an equimolar
ratio and hybridizing to different positions along the CLN2
mRNA (see Note 1) (ttgatgacgagtcccatacg, cggatagtagtccggtttag, attctgcattagatagctca, ttcttgcagcatttcgaagt, aacattggtggagatttctt, gctggtctattagttttgga, taatgttggaccttgtttcc, ccacagacagctcgaacaaa, ataccatttgtcactcgagt, ctcttggaacaatagcggtc,
acaaccaatttggcttggtc, agccaaccagagacaagtag, atgatgtgattacaaccgcc, ccagtagggatgactacatt, gggttgggaccataaaatct, cagagagtcgaggtatacgt, gaccatcaccacagtaatga, gtctagtatatgtctttcca,
gactgacgtttttcagagca, tctacagtggcatcactatc, tttaagtcttcttcttcttc,
ctaagtaagtcgtactgcca, gagaatatgccgtgcgatac, aaaggaccgtggtcttgatt, gctttctgatgtcattggag, atgccgttcattaaggtact, cttccatcaaggagttagga, agaacaccattgaccgtttt, caagtgatattctttcact, gttggatgcaatttgcagtt, gatatggtaagctttctcga, ttcgaaagagcatgatgggg,
gcgaaggaatggatgtgcta, gagtgtggctttgagatgag, atcagagagtgagctcatgt, catattccggctgaaaacgc, cttggagtgattggtgatga, ctgctgaccaaattggtaca, gtgctaccacatatactgtt, ttcaccagactattcacact,
tttgttcgtagatcctttgt, atcattggttgcgttattgc, ttggttttccttgttagact,
attgaggtaatgcgccgttg, ggggaacattccatggttaa, ctatttatggtcccagttgg, gatgaggcactgctagattt, ggtattgcccataccaaaag). Resuspend the probe mix in TE buffer to a final concentration of
25 μM and store in aliquots protected from light at À20
C.
23. Competitor DNA/RNA: 10 mg/mL Sheared salmon sperm
DNA, 10 mg/mL E. coli tRNA. Store at À20
C.
24. Vacufuge: To lyophilize smFISH probes.
25. 100 mm and 150 mm petri dishes.
26. Kimtech tissues.
27. 20Â Saline sodium citrate (SSC) buffer: 3 M NaCl, 0.3 M
sodium citrate-HCl, pH 7.0.
28. smFISH pre-hybridization solution: 10% Formamide, 2Â SSC.
29. Solution F: 20% Formamide, 10 mM NaHPO 4 , pH 7.5. Prepare fresh.
30. Solution H: 4Â SSC, 2 mg/ml BSA, 10 mM VRC. Prepare
fresh.
31. 2Â SSC: Dilute 20Â SSC 1:10 with DDW. Store at room
temperature.
32. 1Â SSC: Dilute 20Â SSC 1:20 with DDW. Store at room
temperature.
33. 10% Formamide/2Â SSC solution: Prepare fresh.
34. 2Â SSC/0.1% Triton X-100 solution.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
55
store in aliquots protected from light at À20
C.
22. CLN2-Q570 probe set: smFISH probes mixed in an equimolar
ratio and hybridizing to different positions along the CLN2
mRNA (see Note 1) (ttgatgacgagtcccatacg, cggatagtagtccggtttag, attctgcattagatagctca, ttcttgcagcatttcgaagt, aacattggtggagatttctt, gctggtctattagttttgga, taatgttggaccttgtttcc, ccacagacagctcgaacaaa, ataccatttgtcactcgagt, ctcttggaacaatagcggtc,
acaaccaatttggcttggtc, agccaaccagagacaagtag, atgatgtgattacaaccgcc, ccagtagggatgactacatt, gggttgggaccataaaatct, cagagagtcgaggtatacgt, gaccatcaccacagtaatga, gtctagtatatgtctttcca,
gactgacgtttttcagagca, tctacagtggcatcactatc, tttaagtcttcttcttcttc,
ctaagtaagtcgtactgcca, gagaatatgccgtgcgatac, aaaggaccgtggtcttgatt, gctttctgatgtcattggag, atgccgttcattaaggtact, cttccatcaaggagttagga, agaacaccattgaccgtttt, caagtgatattctttcact, gttggatgcaatttgcagtt, gatatggtaagctttctcga, ttcgaaagagcatgatgggg,
gcgaaggaatggatgtgcta, gagtgtggctttgagatgag, atcagagagtgagctcatgt, catattccggctgaaaacgc, cttggagtgattggtgatga, ctgctgaccaaattggtaca, gtgctaccacatatactgtt, ttcaccagactattcacact,
tttgttcgtagatcctttgt, atcattggttgcgttattgc, ttggttttccttgttagact,
attgaggtaatgcgccgttg, ggggaacattccatggttaa, ctatttatggtcccagttgg, gatgaggcactgctagattt, ggtattgcccataccaaaag). Resuspend the probe mix in TE buffer to a final concentration of
25 μM and store in aliquots protected from light at À20
C.
23. Competitor DNA/RNA: 10 mg/mL Sheared salmon sperm
DNA, 10 mg/mL E. coli tRNA. Store at À20
C.
24. Vacufuge: To lyophilize smFISH probes.
25. 100 mm and 150 mm petri dishes.
26. Kimtech tissues.
27. 20Â Saline sodium citrate (SSC) buffer: 3 M NaCl, 0.3 M
sodium citrate-HCl, pH 7.0.
28. smFISH pre-hybridization solution: 10% Formamide, 2Â SSC.
29. Solution F: 20% Formamide, 10 mM NaHPO 4 , pH 7.5. Prepare fresh.
30. Solution H: 4Â SSC, 2 mg/ml BSA, 10 mM VRC. Prepare
fresh.
31. 2Â SSC: Dilute 20Â SSC 1:10 with DDW. Store at room
temperature.
32. 1Â SSC: Dilute 20Â SSC 1:20 with DDW. Store at room
temperature.
33. 10% Formamide/2Â SSC solution: Prepare fresh.
34. 2Â SSC/0.1% Triton X-100 solution.
Detection of mRNA and Proteins in Single Cells by smFISH-IF
55