Antisense riboprobes were synthesized and in situ hybridization was performed as previously described [29 (link)], with the addition of 5% dextran sulfate (Sigma-Aldrich D8906) to the hybridization buffer. To increase visualization of staining, some 24 hpf embryos were also incubated in 3%H2O2/0.5%KOH to remove pigmentation. Post-hybridization washes were carried out using a Biolane HTI in situ machine (Huller and Huttner AG). After staining, embryos were stored in MeOH and cleared in 70% glycerol for dissection and imaging. For sectioning, embryos were post-fixed in 4% paraformaldehyde for 1–2 hours at room temp, washed in PBS, and cryoprotected in 30% sucrose in PBS. Embryos were then embedded in OCT and sectioned at 20μm thickness on a Leica cryostat. Images were acquired using an Olympus BX51WI compound microscope and an Olympus Microfire camera. Digital images were cropped and aligned using Adobe Photoshop.
D8906
Manufactured by Merck Group
D8906 is a laboratory equipment product. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
R4251, by Merck Group (2 mentions)
Am9342, by Thermo Fisher Scientific (2 mentions)
Vanadyl robonucleoside complex, by New England Biolabs (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Bx51wi compound microscope, by Olympus (1 mentions)
Microfire camera, by Olympus (1 mentions)
Cryostat, by Leica camera (1 mentions)
Orca r2 ccd camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
E coli trna, by Merck Group (1 mentions)
Am9763, by Thermo Fisher Scientific (1 mentions)
Quasar 570, by Biosearch Technologies (1 mentions)
R8759, by Merck Group (1 mentions)
Stellaris rna, by Biosearch Technologies (1 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (1 mentions)
Bp337 100, by Thermo Fisher Scientific (1 mentions)
Am2616, by Thermo Fisher Scientific (1 mentions)
Stellaris probe, by Biosearch Technologies (1 mentions)
Formamide, by Thermo Fisher Scientific (1 mentions)
Formamide, by Merck Group (1 mentions)
7 protocols using d8906
1
In Situ Hybridization and Embryo Imaging
2
Single Molecule RNA FISH in Neurons
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Neurons were plated on PDL-coated 28-mm glass bottom dishes (Invitrosci) and fixed 7 days later with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized with cold 70% ethanol overnight at 4°C and washed the following morning for 5 minutes with wash buffer (25% formamide, 2X SSC). Neurons were stained with 40 20-nucleotide-long fluorescently labeled (Alexa-647) oligonucleotides designed against the Aag transcript overnight at 37°C in a heavily humidified chamber in hybridization buffer (100 mg/mL dextran sulfate, Sigma, D8906: 0.5 mg/mL E.coli tRNA, Sigma, R4251: 0.5 mg/mL ssDNA, Sigma D9156: 1 mg/mL Ultapure BSA, Ambion, AM2616: 10 mM VRC, New England Biolabs, S1402S: 25% formamide, Ambion, AM9342: 2X SSC, Ambion, AM9763). Finally, cells were stained with DAPI (2 μg/mL) in wash buffer for 30 minutes at 37°C before imaging. All the previous steps were done in nuclease-free solutions to avoid RNA degradation. We occasionally see faint transcripts in Aag-/- cells due to initiation of endogenous transcription before hitting the inserted cassette that disrupts the gene.
Images were taken with a Hamamatsu ORCA-R2 CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].
Images were taken with a Hamamatsu ORCA-R2 CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].
3
Quantifying TAHRE Transcripts in Drosophila Ovaries
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Custom Stellaris FISH probes were designed against TAHRE transcripts by using the Stellaris RNA FISH probe designer version 4.1 (Biosearch Technologies, Inc.; available online at http://www.biosearchtech.com/stellarisdesigner ). The wild-type, p53−, and p53Rescue ovaries were hybridized with the TAHRE Stellaris RNA FISH probe set labeled with Quasar 570 (Biosearch Technologies, Inc.) following the manufacturer's instructions (available online at http://www.biosearchtech.com/stellarisprotocols ). Briefly, ovaries were dissected into PBS and fixed for 45 min at room temperature with 4% formaldehyde solution in PBS. After fixation, ovaries were placed in 70% EtOH overnight at 4°C. The following day, the EtOH was aspirated, and wash buffer (2× SSC, 10% deionized formamide in nuclease-free water) was added for 5 min. The probe was diluted at a concentration of 50 nM in hybridization buffer (2× SSC, 10% dextran sulfate [Sigma, D8906], 1 mg/mL tRNA [Sigma, R8759], 2 mM vanadyl robonucleoside complex [New England Biolabs], 10% deionized formamide in nuclease free water). The wash buffer was aspirated, and the hybridization + probe solution was added to each sample and placed for 24 h at 37°C. The samples were then washed with wash buffer twice for 30 min each at 37°C. VectaShield (Vector Laboratories) with DAPI was added before mounting and imaging.
4
Optimizing Formamide Concentration for RNA Detection
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Following (Raj et al., 2006 (link)), a range of
formamide concentrations was initially tested to empirically determine the optimal
value. 20% (w/v) formamide gave the best results in that it was high enough so
that background noise due to non-specific binding was low, while still low enough
so that the fluorescence signal from target mRNA molecules was not impaired.
10 ml of wash solution contains 1.76 ml of formamide (Ambion, AM9342), 1 ml of 20×
SSC (Ambion, AM9763), and 10 μl Tween-20 (Fisher Scientific, BP337-100). Wash
solution was made fresh and stored on ice until use. 10 ml of hybridization
solution contains 1 g of dextran sulfate (Sigma, D8906), 1.76 ml of formamide, 10
mg of E. coli tRNA (Sigma, R4251), 1 ml of 20× SSC, 40 μl of 50
mg/ml BSA (Ambion, AM2616), and 100 μl of 200 mM ribonucleoside vanadyl complex
(New England Biolabs, Ipswich, NY, S1402S). Hybridization solution was filter
sterilized and aliquots of 500 μl were stored at -20°C.
formamide concentrations was initially tested to empirically determine the optimal
value. 20% (w/v) formamide gave the best results in that it was high enough so
that background noise due to non-specific binding was low, while still low enough
so that the fluorescence signal from target mRNA molecules was not impaired.
10 ml of wash solution contains 1.76 ml of formamide (Ambion, AM9342), 1 ml of 20×
SSC (Ambion, AM9763), and 10 μl Tween-20 (Fisher Scientific, BP337-100). Wash
solution was made fresh and stored on ice until use. 10 ml of hybridization
solution contains 1 g of dextran sulfate (Sigma, D8906), 1.76 ml of formamide, 10
mg of E. coli tRNA (Sigma, R4251), 1 ml of 20× SSC, 40 μl of 50
mg/ml BSA (Ambion, AM2616), and 100 μl of 200 mM ribonucleoside vanadyl complex
(New England Biolabs, Ipswich, NY, S1402S). Hybridization solution was filter
sterilized and aliquots of 500 μl were stored at -20°C.
5
Stellaris RNA FISH Probe Detection
6
Single Molecule FISH Assay for FMR1
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Plated wild-type S2 cells were first treated with KRB (see above), and fixed and labeled for endogenous FMR1 or Sec16 as described above in the immunofluorescence section. After incubation with the secondary antibody, cells were washed three times with PBS and cells were post-fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min. Following a washing three times in PBS, cells were further incubated for 5 min in 10% formamide (17899, Thermo Fisher Scientific) in DEPC-treated water. They were then incubated overnight on a droplet containing one fluorescent smFISH probe [125 nM in 1% dextransulfate (D8906, Sigma-Aldrich), 10% formamide in DEPC-treated water at 37°C] in a moistened chamber to avoid drying. Cells were washed twice for 30 min with 10% formamide in DEPC-treated water and mounted with Prolong antifade medium (plus DAPI) on a microscope slide. The TMR-oligo(dT)30× was purchased from IDT. A widefield Leica MM-AF microscope was used with a 100× lens for imaging.
7
Fluorescence In Situ Hybridization Brains
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After fixation, permeabilization and O/N incubation with GFP booster (description below), brains were washed 3x15min in PBT3 and fixed a second time 30 min in 4%PFA. Then, brains were rinse 3x in PBS, washed 1x5 min in 2xSSCT (2X Saline Sodium Citrate (Euromedex #EU0300-A) 0,1% Tween-20 (Sigma Aldrich #P1379) diluted in water), 1x5 min in 2xSSCT/50% formamide (Sigma Aldrich #47671), transferred in pre-warmed 2xSSCT/50% formamide and pre-hybridized 3 min at 92°C. In the meantime, DNA probes diluted in the Hybridization Buffer (20%dextransulfate (Sigma Aldrich #D8906), 2xSSCT, 50%formamide, 0,5mg/ml salmon DNA sperm (Sigma Aldrich #D1626)) were denature at 92°C. After removal of the supernatant, brains were incubated in the probes solutions and hybridize 5 min at 92°C and O/N at 37°C. Brains were then rinse at RT, washed 1x10 min at 60°C and 1x5 min at RT in 2xSSCT. Finally, after a rinse in PBS brains were mounted as described below.
DNA probes used in this study were against chromosomes X (80ng/µl), II (40ng/µl) and III (80ng/µl).
DNA probes used in this study were against chromosomes X (80ng/µl), II (40ng/µl) and III (80ng/µl).
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