M-FISH experiments were performed with fresh-frozen brain tissue. Briefly, P21 male and female Opn5cre/+; Ai14 mice were sacrificed and their brains rapidly dissected into cryo-embedding medium. Embedded brains were snap-frozen in liquid nitrogen, and 14 μm cryosections of the POA were obtained and processed for M-FISH using the RNAscope® Fluorescent Multiplex Reagent Kit V1 (ACDBio). Probes against the following mRNAs were used: Slc32a1 (Vgat), Slc17a6 (Vglut2), Adcyap1 (PACAP), Bdnf (BDNF), and tdTomato. In situ hybridization was performed as per the manufacturer’s protocol for fresh frozen tissue. Briefly, POA sections were pretreated by serial immersion of the slides in 1x PBS, nuclease-free water, and 100% EtOH at room temperature for two minutes each. Probe hybridization was achieved by incubating sections in 40 μL of mRNA target probes for 2 hours at 40°C, followed by signal amplification using manufacturer-provided Amp1, Amp2, Amp3, and Amp4 reagents for 30, 15, 30, and 15 minutes respectively at 40°C. Each incubation step was followed by two 2-min washes of manufacturer-provided wash buffer. Slides were mounted using Tris-buffered Fluoro-Gel mounting medium (Electron Microscopy Sciences).
Tris buffered fluoro gel mounting medium
Manufactured by Electron Microscopy Sciences
Tris-buffered Fluoro-Gel is a mounting medium designed for use with fluorescence microscopy. It is a water-based, glycerol-based gel that helps preserve and protect fluorescent samples during long-term storage and imaging.
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3 protocols using tris buffered fluoro gel mounting medium
1
Multiplexed RNA Detection in Mouse Brain
2
Multiplexed RNA Detection in Mouse Brain
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M-FISH experiments were performed with fresh-frozen brain tissue. Briefly, P21 male and female Opn5cre/+; Ai14 mice were sacrificed and their brains rapidly dissected into cryo-embedding medium. Embedded brains were snap-frozen in liquid nitrogen, and 14 μm cryosections of the POA were obtained and processed for M-FISH using the RNAscope® Fluorescent Multiplex Reagent Kit V1 (ACDBio). Probes against the following mRNAs were used: Slc32a1 (Vgat), Slc17a6 (Vglut2), Adcyap1 (PACAP), Bdnf (BDNF), and tdTomato. In situ hybridization was performed as per the manufacturer’s protocol for fresh frozen tissue. Briefly, POA sections were pretreated by serial immersion of the slides in 1x PBS, nuclease-free water, and 100% EtOH at room temperature for two minutes each. Probe hybridization was achieved by incubating sections in 40 μL of mRNA target probes for 2 hours at 40°C, followed by signal amplification using manufacturer-provided Amp1, Amp2, Amp3, and Amp4 reagents for 30, 15, 30, and 15 minutes respectively at 40°C. Each incubation step was followed by two 2-min washes of manufacturer-provided wash buffer. Slides were mounted using Tris-buffered Fluoro-Gel mounting medium (Electron Microscopy Sciences).
3
Multiplex Fluorescence in situ Hybridization of Opn5-expressing Neurons
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M-FISH experiments were performed as previously described [6 (link)]. Briefly, P42-49 male and female Opn5cre;Gnasfl/fl;Ai14 (mutant) and Opn5cre;Gnasfl/+;Ai14 or Opn5cre;Gnas+/+;Ai14 (control) mice were euthanized by isoflurane and brains were rapidly dissected and snap-frozen in liquid nitrogen. 14-micron cryosections were obtained and processed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD Bio). POA sections were fixed in ice-cold RNase-free paraformaldehyde for 1 hour and pre-treated in 1X RNase-free PBS and 50%, 75%, and 100% ethanol for five minutes each. After treatment with hydrogen peroxide for 15 minutes and protease for 30 minutes at 22°C, sections were incubated in target probes against Gnas Exon 1 (Probe-Mm-Gnas-O1-C2, Cat No. 526891-C2) (1:50) and tdTomato (Probe-tdTomato-C3) (1:100) at 40°C for 2 hours. Signal amplification using manufacturer-provided Amp1, Amp2, and Amp3 reagents for 30, 30, and 15 minutes was performed at 40°C, with 4-minute washes of 1X wash buffer in between. C2 and C3 were separately amplified with TSA Vivid Fluorophores 520 and 570 (1:1500 each), respectively. DAPI was stained for 15 seconds. Slides were then mounted using Tris-buffered Fluoro-Gel mounting medium (Electron Microscopy Sciences).
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