Fresh fecal pellets were collected from 12 conventional 5-month-old C57BL/6 mice obtained from the Jackson Laboratory. To remove interindividual variation as a variable, the pellets were pooled and homogenized using a spatula to make a fecal master mix. The master mix was split into aliquots of 8 mg each. The aliquots were either resuspended in 200 μl of a preservation solution and stored at room temperature (∼22°C) in the dark or were flash-frozen using liquid nitrogen and stored at −80°C. The preservation treatments are discussed in detail below. In brief, we tested six preservation treatments: flash-freezing in liquid nitrogen followed by storage at −80°C (FF treatment); preservation at room temperature in RNAlater (RNAlater stabilization solution, Invitrogen; R treatment); a combination of RNAlater and flash-freezing (RF treatment); an RNAlater-like preservation buffer (NAP buffer) as described by Menke et al. (59 (link)) (N treatment); the same NAP buffer autoclaved (AN treatment); and 95% ethanol (E treatment). We tested the effectiveness of the treatments at preserving microbiome samples over two storage durations: 1 and 4 weeks. We prepared four replicate samples per treatment and time point, with each replicate being an 8-mg aliquot of the fecal master mix described above.
Rnalater stabilization
Manufactured by Thermo Fisher Scientific
RNAlater stabilization solution is a reagent used to stabilize and preserve RNA in biological samples. It immediately stabilizes the RNA, preventing its degradation, and allows for the safe storage and transportation of samples without the need for freezing. The solution can be easily applied to a variety of sample types, including tissues, cells, and biofluids.
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4 protocols using rnalater stabilization
1
Preserving Gut Microbiome Samples
2
Preserving Gut Microbiome Samples
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Fresh fecal pellets were collected from 12 conventional 5-month-old C57BL/6 mice obtained from the Jackson Laboratory. To remove interindividual variation as a variable, the pellets were pooled and homogenized using a spatula to make a fecal master mix. The master mix was split into aliquots of 8 mg each. The aliquots were either resuspended in 200 μl of a preservation solution and stored at room temperature (∼22°C) in the dark or were flash-frozen using liquid nitrogen and stored at −80°C. The preservation treatments are discussed in detail below. In brief, we tested six preservation treatments: flash-freezing in liquid nitrogen followed by storage at −80°C (FF treatment); preservation at room temperature in RNAlater (RNAlater stabilization solution, Invitrogen; R treatment); a combination of RNAlater and flash-freezing (RF treatment); an RNAlater-like preservation buffer (NAP buffer) as described by Menke et al. (59 (link)) (N treatment); the same NAP buffer autoclaved (AN treatment); and 95% ethanol (E treatment). We tested the effectiveness of the treatments at preserving microbiome samples over two storage durations: 1 and 4 weeks. We prepared four replicate samples per treatment and time point, with each replicate being an 8-mg aliquot of the fecal master mix described above.
3
Tau Protein Immunogold Labeling in Mouse Retina
4
Tau Protein Immunogold Labeling in Mouse Retina
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Mouse retinas stored in RNAlater stabilization solution (Invitrogen, AM7020) were homogenized in 1× RIPA buffer (Cell Signaling) containing protease inhibitor cocktail and PMSF. Formvar-carbon coated copper grid were fixed with 2% PFA and 1% glutaraldehyde for 5 min then incubated in 10 μl of retinal homogenate for 20 min at room temperature. Grids were washed once with 0.01 M PBS and 50 mM glycine for 3 min, then blocked in 1% cold-water fish skin gelatin in PBS for 1 h. The grids were incubated in anti-3RTau antibody cocktail in 1% cold-water fish skin gelatin (1:50, Millipore) for 2 h at room temperature. After washing six times with PBS for 3 min, the grids were transferred to drops of 1% cold-water fish skin gelatin containing anti-mouse gold-conjugated antibodies (1:100, Sigma-Aldrich) and incubated for 1 h. Grids were washed six times with PBS for 3 min, counterstained with 3% (w/v) uranyl acetate for 5 min, then washed with double distilled water and blotted dry. Protein content was visualized using a JEOL 1200 EX II transmission electron microscope.
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