perm/ wash buffer kit  (Becton Dickinson)

Journal: bioRxiv

Article Title: HIF-1α coordinates adrenal steroidogenesis through direct transcriptional control and regulation of miRNA biogenesis

doi: 10.64898/2026.02.24.707817

Figure Lengend Snippet: A) Schematic representation of the CUT&Tag protocol. Under standard culture conditions (21% O□), Y-1 cells express undetectable levels of HIF-1α protein. For the CUT&Tag procedure, the cells were either left untreated as a control or treated with DMOG for 24 hours to stabilize HIF-1α. This treatment leads to nuclear accumulation of HIF-1α and its binding to target genes. The nuclei were then isolated, lightly fixed with formaldehyde, and bound to concanavalin A-coated beads. The HIF-1α-bound chromatin regions were labeled on the beads using a primary anti-HIF-1α antibody, a secondary antibody, and protein A-Tn5 adapters. The tagged DNA fragments were then released, PCR-amplified, and subjected to Illumina next-generation sequencing. B) The genomic distribution of enriched (left) and depleted (right) HIF-1α-bound sequences in DMOG-treated versus control cells (see also Additional file 2). C) Top 15 KEGG pathways enriched among HIF-1α-bound genes. Bar length indicates the pathway enrichment ratio, and colors represent FDR (see also Additional file 3). D) Motif enrichment analysis of HIF-1α-bound sequences revealed conserved binding motifs for HIF-1/2α and 30 additional transcription factors (p < 0.01, Benjamini < 0.1) that are grouped into six major families (see Additional file 4).

Article Snippet: The beads were then washed with 0.5 ml of CUT&Tag wash buffer and the pA-Tn5 adapter complex (Epicypher, #15-1117) in 300 wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor EDTA-Free tablets; Merck, #11836170001) was added at 1:20.

Techniques: Control, Binding Assay, Isolation, Labeling, Amplification, Next-Generation Sequencing

Journal: bioRxiv

Article Title: HIF-1α coordinates adrenal steroidogenesis through direct transcriptional control and regulation of miRNA biogenesis

doi: 10.64898/2026.02.24.707817

Figure Lengend Snippet:

Article Snippet: The beads were then washed with 0.5 ml of CUT&Tag wash buffer and the pA-Tn5 adapter complex (Epicypher, #15-1117) in 300 wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor EDTA-Free tablets; Merck, #11836170001) was added at 1:20.

Techniques:

Journal: bioRxiv

Article Title: HIF-1α coordinates adrenal steroidogenesis through direct transcriptional control and regulation of miRNA biogenesis

doi: 10.64898/2026.02.24.707817

Figure Lengend Snippet: A) Schematic depiction of the miRNA processing pathway. The pathway components identified in the HIF-1α CUT&Tag assay are depicted with protein names highlighted in red. Among the genes bound by HIF-1α are both members of the nuclear microprocessor complex ( Drosha and Dgcr8 ) as well as a number of RISC components ( Ago1-4 , Snd1 , Mtdh , Tarbp1 and Tnrc6a/b ). B) Identified miRNA biogenesis and function genes with the assigned location of the putative HIF-1α binding region and a number of canonical HIF-1α binding sites detected within each region.

Article Snippet: The beads were then washed with 0.5 ml of CUT&Tag wash buffer and the pA-Tn5 adapter complex (Epicypher, #15-1117) in 300 wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor EDTA-Free tablets; Merck, #11836170001) was added at 1:20.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Sox8 and Sox9 regulate differentiation and nuclear positioning of retinal Müller glia

doi: 10.64898/2026.01.12.698638

Figure Lengend Snippet: (A) Schematic of the CUT&RUN experimental pipeline. (B) Overlap of P2 Sox8/9 peaks and P2 RPC DARs. (C) GO enrichment of P2 Sox8/9 peaks and P2 RPC DARs. (D) Distribution of Sox8/9 binding loci in distinct gene regions at P2 and P17 compared to H3K27ac and H3K4me3 controls . (E) Distribution of Sox8/9 binding loci relative to transcription start site (TSS) at P2 and P17 compared to H3K27ac and H3K4me3 controls . (F) Number of Sox8/9 CUT&RUN peaks that overlap with enriched/differentially accessible regions in either P2 RPCs, P17 Control MGs, or P17 Sox8/9 cKO MGs. (G) Percentage of Sox8/9 CUT&RUN peaks that overlap with enriched/differentially accessible regions in either P2 RPCs or P17 Control MGs. (H) GO enrichment of overlapping Sox8/9 DKO DEG, DAR, and P17 Sox8/9 peaks.

Article Snippet: Cold CUT&RUN wash buffer was prepared according to manufacturer’s guidance (EpiCypher, 14-1048) and was used as substitute for the final nuclei wash and resuspension.

Techniques: Binding Assay, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Tmprss3 is expressed in several cell types of the inner ear including type II but hardly in type I spiral ganglion neurons

doi: 10.3389/fncel.2025.1690227

Figure Lengend Snippet: Tmprss3 expression in wholemount organs of Corti. (A) Representative images of Tmprss3 mRNA and protein expression in the apical part of the organ of Corti of a P32 wildtype ( Tmprss3 +/+ ) and a P10 heterozygous ( Tmprss3 + ⁣/− ) mouse. The organs of Corti are immunostained with an anti-TMPRSS3 antibody (TMPRSS3 Ab, magenta) and the hair cell marker calretinin (blue). Tmprss3 mRNA is labeled with an RNAscope probe (yellow). TMPRSS3 protein is localized in hair bundles of inner and outer hair cells and in the spiral limbus, while Tmprss3 mRNA is expressed in the corresponding cell bodies. Scale bars: 50 μm. (B) High magnification of immunolabeling for TMPRSS3 (magenta) and calretinin (blue), depicting the stereocilia of outer (left) and inner hair cells (right), indicating strong presence of TMPRSS3 in stereocilia of mature hair cells (upper panel) and weaker staining of immature hair cells of Tmprss3 + ⁣/− mice (middle panel). Lower panel: control of immunostaining performed on homozygous knock-out ( Tmprss3 –/– ) tissue at P10, where no immunolabeling of stereocilia occured. Scale bars: 5 μm.

Article Snippet: Subsequently, the slides were washed in 1× RNAscope Wash Buffer (used for all subsequent washing steps), and 3 amplification steps were performed using RNAscope Amp 1, Amp 2 and Amp 3 (323110, ACD Bio-Techne) and followed by washing after each step.

Techniques: Expressing, Marker, Labeling, RNAscope, Immunolabeling, Staining, Control, Immunostaining, Knock-Out

Journal: Frontiers in Cellular Neuroscience

Article Title: Tmprss3 is expressed in several cell types of the inner ear including type II but hardly in type I spiral ganglion neurons

doi: 10.3389/fncel.2025.1690227

Figure Lengend Snippet: Representative images of Tmprss3 mRNA via RNAscope detection (yellow) and calretinin immunostaining (magenta) in different developmental stages of the murine cochlea. Overview of a mid-modiolar cryosections of the cochlea from wildtype mice aged P8 (upper) , P14 (middle) and P29 (lower panel) . Tmprss3 mRNA is expressed in both inner and outer hair cells, in supporting cells, in the spiral limbus (SL), cells of lateral wall, and in SGNs. Calretinin immunostaining labels hair cells and most of the type I SGNs. Scale bars: 100 μm.

Article Snippet: Subsequently, the slides were washed in 1× RNAscope Wash Buffer (used for all subsequent washing steps), and 3 amplification steps were performed using RNAscope Amp 1, Amp 2 and Amp 3 (323110, ACD Bio-Techne) and followed by washing after each step.

Techniques: RNAscope, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Tmprss3 is expressed in several cell types of the inner ear including type II but hardly in type I spiral ganglion neurons

doi: 10.3389/fncel.2025.1690227

Figure Lengend Snippet: Representative images of an RNAscope experiment to visualize Tmprss3 mRNA expression (yellow) in the lateral wall. To identify root cells, RNAscope was employed for detecting Kcnj10 mRNA (blue). DAPI staining of cellular nuclei (white), was added to illustrate the tissue structure. (A) Root cells (arrows) of the lateral wall, positive for Kcnj10 mRNA, from a mid-modiolar cochlear cryosection from a wildtype premature (P8) and mature (P29) mouse cochlea, also express Tmprss3 mRNA. Scale bars: 50 μm. (B) Tmprss3 mRNA expression in root cells is higher in apical turns (*) compared to midbasal turns (**). Scale bars: 100 μm. Arrows: root cells; IHC, inner hair cells; OHC, outer hair cells.

Article Snippet: Subsequently, the slides were washed in 1× RNAscope Wash Buffer (used for all subsequent washing steps), and 3 amplification steps were performed using RNAscope Amp 1, Amp 2 and Amp 3 (323110, ACD Bio-Techne) and followed by washing after each step.

Techniques: RNAscope, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Tmprss3 is expressed in several cell types of the inner ear including type II but hardly in type I spiral ganglion neurons

doi: 10.3389/fncel.2025.1690227

Figure Lengend Snippet: Tmprss3 expression in type I and II SGNs at different developmental stages. (A) Representative image of modiolar cryosection displaying Tmprss3 mRNA expression (yellow) in SGNs in type I (Calret +) and type II (Th +) SGNs at the age of P8 (upper), P14 (middle) and P29 (lower panel). While at P8 and P14, Tmprss3 mRNA is found rather uniformly distributed across the Rosenthal’s canal, the mRNA of Tmprss3 becomes restricted to few cells at P29, which were typically Th + cells. Scale bars: 50 μm. (B) Higher magnification images from insets in A, Scale bars: 20 μm. (C) TMPRSS3 immunostaining (magenta) and mRNA labeling with RNAscope (yellow) in SGNs at age P32 in a wholemount spiral ganglion, combined with calretinin immunostaining (blue) for type I SGNs. Scale bars: 20 μm.

Article Snippet: Subsequently, the slides were washed in 1× RNAscope Wash Buffer (used for all subsequent washing steps), and 3 amplification steps were performed using RNAscope Amp 1, Amp 2 and Amp 3 (323110, ACD Bio-Techne) and followed by washing after each step.

Techniques: Expressing, Immunostaining, Labeling, RNAscope