probe wash  (Thermo Fisher)

Journal: Nature Aging

Article Title: Multi-tissue transcriptomic aging atlas reveals predictive aging biomarkers in the killifish

doi: 10.1038/s43587-026-01074-6

Figure Lengend Snippet: a , Spearman’s rank correlation ( ρ ) heat maps for the genes upregulated (left) or downregulated (right) with age shared across at least six tissues. Gray box indicates that Spearman’s correlation was not calculated because the expression level of a particular gene was lower than the expression threshold (transcripts per million (TPM) > 0.5 in over 80% of samples). Killifish-specific genes are named after their gene locus numbers (for example, LOC107378024 ) or are designated in lowercase letters. When a killifish gene has a human ortholog, the human gene name is used and shown in uppercase letters. b , Hypergeometric GO enrichment results for the genes upregulated (top) or downregulated (bottom) with age that are shared across at least five tissues. Enrichment significance was assessed using a hypergeometric test implemented in GOstats, with the background (‘universe’) defined as all genes with non-NA (not available) FDR-adjusted P values for the corresponding comparison. P values were adjusted for multiple hypothesis testing using the Benjamini–Hochberg method, and GO terms with FDR < 0.05 were considered significant. Dot size represents −log 10 of the FDR-adjusted P value (that is, FDR after multiple hypothesis testing). c , Representative maximum z -projected hybridization chain reaction (HCR; RNA in situ) images for ncRNA-3777 mRNAs in male and female guts, at young (57–60 days) and old (120–130 days) ages. Scale bar, 5 µm. d , Quantification of HCR images as the average number of ncRNA-3777 transcripts per cell. Here and after, one individual fish represented an independent biological replicate. Each dot is a fish; n = 4 fish per condition. In-graph statistical comparisons were performed using a two-sided Mann–Whitney U -test between the indicated groups. Below-graph statistics were assessed using a two-way analysis of variance (ANOVA) including age, sex and the age–sex interaction as factors. e , Normalized RNA-seq expression values (DESeq2-normalized counts) in the gut for the ncRNA-3777 gene are shown for individual animals across age bins and sex. Dots represent expression values from individual fish. Bars indicate the mean expression for each sex and age bin, and error bars denote ± s.e.m., calculated across biological replicates within each group. Age bins correspond to defined ranges of days after hatching and are color coded as indicated. f , Representative maximum z -projected HCR (RNA in situ) images for IGF2BP3 with conditions as described in c . g , h , Quantification and statistics were performed as in d and e , respectively, for IGF2BP3 . i , z -scaled locally estimated scatterplot smoothing (LOESS) regression fits of the gene expression trajectories across age for the genes ncRNA-3777 and IGF2BP3 . j , Age-associated gene expression change (log 2 -fold change) in mouse and killifish brain for the genes in the ‘Common Aging Score’ gene set, which is a published brain-wide gene signature of aging defined in Hahn et al. . Differential expression was performed using DESeq2, comparing 133–134 days and 47–52 days in killifish (corresponding to ~30% and ~100% colony survival, respectively) or between 27 months and 3 months in mice (corresponding to 50–75% and 100% colony survival, respectively). Statistical significance was assessed using the two-sided Wald test implemented in DESeq2, and P values were adjusted for multiple hypothesis testing using the Benjamini–Hochberg method. The 13 of 82 mouse genes with significant age-associated change in the killifish are shown (FDR-adjusted P value < 0.05). Green denotes a mouse; pink denotes a killifish. The mouse data are from Schaum et al. .

Article Snippet: After prehybridization, the buffer was removed, and 100 μl hybridization buffer (for each HCR probe, use 1 μl of the 0.5 pmol μl −1 stock per 100 μl hybridization buffer) was added to each slide, followed by a 37 °C incubation for 16–20 h. After hybridization, each slide was washed with 500 μl HCR probe wash buffer (Molecular Instruments, buffer type: tissue section), 500 μl 75% wash buffer (75% HCR probe wash buffer, 25% 5× SSCT), 500 μl 50% wash buffer (50% HCR probe wash buffer, 50% 5× SSCT), 500 μl 25% wash buffer (25% HCR probe wash buffer, 75% 5× SSCT) and 500 μl 5× SSCT (diluted from 20× SSCT (Ambion, AM9770) with nuclease-free water) at 37 °C with a 15-min incubation for each wash. Next, each slide was incubated in 200 μl HCR amplification buffer (Molecular Instruments, buffer type: tissue section) for 30 min to 4 h before switching to 100 μl amplification buffer supplemented with the fluorescent hairpin pairs (prepared according to the manufacturer’s instructions) for 20–24-h incubation at room temperature in the dark.

Techniques: Expressing, Comparison, Hybridization, In Situ, MANN-WHITNEY, RNA Sequencing, Gene Expression, Quantitative Proteomics