Rnase free bsa

Fluorescent in situ hybridization was performed as described previously [48 (link)].
Briefly, testes were dissected in 1X PBS and then fixed in 4% formaldehyde/PBS for 45 minutes. After fixing, they were rinsed 2 times with 1X PBS, then resuspended in 70% EtOH, and left overnight at 4°C. The next day, testes were washed briefly in wash buffer (2X SSC and 10% deionized formamide), then incubated overnight at 37°C in the dark with 50 nM of Quasar 570 labeled Stellaris probe against dpp mRNA (LGC Biosearch Technologies, a gift from Michael Buszczak [49 (link)]) in the hybridization buffer containing 2X SSC, 10% dextran sulfate (Sigma-Aldrich Inc., St Louis, Missouri), 1 μg/μl of yeast tRNA (Sigma-Aldrich Inc.), 2 mM vanadyl ribonucleoside complex (NEB), 0.02% RNAse-free BSA (ThermoFisher), and 10% deionized formamide. On the third day, testes were washed 2 times for 30 minutes each at 37°C in the dark in the prewarmed wash buffer (2X SSC, 10% formamide) and then resuspended in a drop of VECTASHIELD with DAPI (Vector Lab, H-1200).
For quantification of the FISH signal, z-stacks were collected at 0.5 μm intervals using the same acquisition settings of for confocal microscopy using a Zeiss LSM800. The total number of particles in the hub was counted using Octane1.5.1 (Super-resolution Imaging and Single Molecule Tracking Software (https://github.com/jiyuuchc/Octane)).

5′ end biotin-labeled TP53 3′UTR RNA fragments with different PAS and other mutations and miRNA of interest labeled with biotin at the 3′end were commercially synthesized (Sigma-Aldrich). H1299 cells were seeded 1 day before transfection in 10 -cm tissue culture dish at 50% confluent. Twenty four hours later, cells were transfect with 5′ end biotinylated TP53 3′UTR RNA fragments (final concentration of 100 nM) or 3′ end biotinylated miRNA (final concentration of 100 nM), and/or a plasmid carrying a TP53 3′UTR (10 µg) according to the manufacturer’s guideline. Twenty four hours post transfection, cells were lysed in 550 μl of lysis buffer supplemented with protease inhibitors and RNase inhibitor, followed by incubation on ice for 10 min. The cell lysates were centrifuged at 4 °C, 14,000 g for 10 min, and the supernatant was subjected to Pierce™ Streptavidin Magnetic Beads in the presence of 2% RNase-free BSA (ThermoFisher Scientific) and 2% yeast tRNA (ThermoFisher Scientific) to block nonspecific binding. After 2 h of incubation, the beads were placed on a magnetic stand for 8 min and washed four times with 1 ml of lysis buffer. Purified RNAs were extracted by RNAzol^® RT (RN 190, Molecular Research Center; Cincinnati, OH) and subjected to reverse transcription and quantitative real-time PCR to determine the beads-bound miRNAs or 3′UTRs.

Fluorescent in situ hybridization was performed as described previously [62 ] with some modifications. Briefly, ovaries were dissected in 1X PBS and then fixed in 4% formaldehyde/PBS for 45 minutes at room temperature. After fixiation, ovaries were briefly rinsed 2 times with 1X PBS, then resuspended in ice-cold 70% EtOH, and incubated at 4°C for 2 hours. Then, ovaries were rinsed briefly in wash buffer (2X SSC and 10% deionized formamide), then hybridized for 16 hours at 37°C in the dark with 50 nM of Quasar 570 labeled Stellaris probe against entire nanos 3’UTR sequence or lky full length cDNA sequence (LGC Biosearch Technologies) in the Hybridization Buffer containing 2X SSC, 10% dextran sulfate (MilliporeSigma), 1 μg/μl of yeast tRNA (MilliporeSigma), 2 mM vanadyl ribonucleoside complex (NEB), 0.02% RNase-free BSA (ThermoFisher), and 10% deionized formamide. Then ovaries were washed 2 times for 30 minutes each at 37°C in the dark in the prewarmed wash buffer (2X SSC, 10% formamide) and then resuspended in a drop of VECTASHIELD with DAPI. Samples were placed on nutator for all incubation steps. Images were taken using a Zeiss LSM800 with Airyscan with a 63 ×oil immersion objective (NA = 1.4) and processed using Fiji.

The smFISH technique was used to determine the spatial expression of genes encoding TNFα, IFNγ, and NOS2 on the lung sections as we previously reported [40 (link)]. Briefly, 5 µm lung sections were equilibrated in a wash buffer, containing 10% Formamide in 2× SSC, followed by overnight incubation of the sections at 37 °C in a hybridization solution containing 10% dextran sulfate, 1 mg/mL Escherichia coli tRNA, 2 mM ribonucleoside vanadyl complex (New England Biolabs, Ipswich, MA, USA), 0.02% RNase-free BSA (ThermoFisher Scientific, Waltham, MA, USA), 10% formamide, and 500 ng/mL of fluorescently-labeled probes of the target gene. After hybridization, the slides were washed twice in hybridization wash buffer at room temperature, treated with TrueBlack Lipofuschin autofluorescence quencher, and mounted with coverslips.

Fluorescence in situ hybridization was performed as described previously. Briefly, testes were dissected in 1X PBS and then fixed in 1 ml of 4% formaldehyde/PBS for 45 minutes. Fixed testes were rinsed 2 times with 1 ml of 1X PBS, then resuspended in 1 ml of ice-cold 70% EtOH, and incubated for 1 hour-overnight at 4 °C. Testes were rinsed briefly in 1 ml of wash buffer (2X SSC and 10% deionized formamide), then incubated overnight at 37 °C for 16 hours in the dark with 50 nM of Stellaris probes in 200 µl of Hybridization Buffer containing 2X SSC, 10% dextran sulfate (MilliporeSigma), 1 μg/μl of yeast tRNA (MilliporeSigma), 2 mM vanadyl ribonucleoside complex (NEB), 0.02% RNase-free BSA (ThermoFisher), and 10% deionized formamide. Then, testes were washed 2 times for 30 minutes each at 37 °C in the dark in 1 ml of prewarmed wash buffer (2X SSC, 10% formamide) and resuspended in a drop of VECTASHIELD with DAPI. Quasar 570 labeled Stellaris probe against a third intron sequence of Stat92E gene and Quasar 670 labeled Stellaris probe against a second exon of Stat92E gene were obtained from LGC Biosearch Technologies (target sequences are provided in Supplementary Data 1). Quasar 570 labeled Stellaris probe against the nanos 3’UTR sequence was gift from Michael Buszczak.