spectral dapi  (Akoya Biosciences)

Journal: bioRxiv

Article Title: A spatial single-cell atlas of the claustro-insular region uncovers key regulators of neuronal identity and excitability

doi: 10.1101/2024.11.05.622016

Figure Lengend Snippet: a Representative image of a coronal section (bregma +1.045) of the claustro-insular region labeled by smFISH with probes for Nr4a2 (purple), Syt17 (orange) and Slc17a6 (white). Arrowheads point towards a CLA cell (white) and a shell cell (orange) shown in ( b,c ). The dotted line indicates the border of the external capsule. Scale bar: 200 μm. b,c Higher magnification of cells characterized by high ( b ) and low ( c ) expression of Nr4a2 . DAPI staining of the nuclei (blue). Scale bar: 10 µm.

Article Snippet: Sections were washed twice for 5 minutes in Sample Prep Wash Buffer and incubated for 15 minutes on a rocker with either 3 ml of Verification Staining Reagent (ref. 20300014, Vizgen, for verification samples) or 3 ml of DAPI and PolyT Staining Reagent (ref. 20300021, Vizgen, for experimental samples).

Techniques: Labeling, Expressing, Staining

Journal: bioRxiv

Article Title: A spatial single-cell atlas of the claustro-insular region uncovers key regulators of neuronal identity and excitability

doi: 10.1101/2024.11.05.622016

Figure Lengend Snippet: a,b Images of coronal sections of the CLA from an Nr4a2 wt/wt mouse ( a ) and an Nr4a2 del/wt mouse ( b ) with Nr4a2 mRNA transcripts labeled by smFISH (white). The white square highlights the region magnified in the inset. DAPI staining of the nuclei (blue). Scale bar: 200 μm. c Mean number of Nr4a2 mRNA puncta detected per cell in the claustro-insular area. Each dot represents the mean of all cells from one section of an Nr4a2 del/wt mouse relative to the mean of all sections of Nr4a2 wt/wt mice processed in the same batch, and the bar represents the mean ± SD. n = 4 Nr4a2 wt/wt mice, 26 sections, 55’697 cells; n = 4 Nr4a2 del/wt mice, 34 sections, 71’389 cells. *** p<0.001; chi-square-based LRT applied to a negative binomial GLMM with quadratic parametrization. d Total number of significantly modulated genes in Nr4a2 del/wt mice in each cell type from the scRNAseq dataset. n = 5 Nr4a2 wt/wt mice, n = 8 Nr4a2 del/wt mice. e,f Violin plots illustrating the distribution of expression for downregulated ( e ) and upregulated ( f ) genes across claustro-insular neuronal populations from Nr4a2 wt/wt and Nr4a2 del/wt mice in the scRNAseq dataset (normalized data are shown in log 10 scale). g,h Mean number of mRNA puncta of downregulated ( g ) and upregulated ( h ) genes detected per cell in Nr4a2 + and/or Oprk1 + cells from the claustro-insular region of Nr4a2 wt/wt and Nr4a2 del/wt mice. Each dot represents the mean of cells from one section of an Nr4a2 del/wt mouse relative to the mean of all sections of Nr4a2 wt/wt mice processed in the same batch, and bars represent the mean ± SD. n = 2-3 Nr4a2 wt/wt mice, 4-14 sections; n = 2-3 Nr4a2 del/wt mice, 5-15 sections. * p<0.05, *** p<0.001; chi-square-based LRT applied to a negative binomial GLMM with quadratic parametrization; p -values were adjusted for multiple comparisons using the Holm method. i,j Cell type-specific modulation of Ntm (i) and Ryr2 (j) gene expression. Top : Violin plots at the top of each panel show the distribution of expression of cell marker genes used for cell type identification in the smFISH data (raw data are shown in linear scale). Bottom : Box plots showing the distribution of Ntm (i) and Ryr2 (j) expression levels within the different identified cell populations from Nr4a2 wt/wt and Nr4a2 del/wt mice in the smFISH dataset. Each dot represents the mean expression level of all cells from one section. Box limits represent Q1 to Q3, the line represents the median, error bars represent ±1.5*IQR. n = 2 Nr4a2 wt/wt mice, 6-8 sections, n = 2 Nr4a2 del/wt mice, 6-8 sections. * p<0.05, *** p<0.001; chi-square-based LRT applied to a negative binomial GLMM with quadratic parametrization; p -values were adjusted for multiple comparisons using the Holm method.

Article Snippet: Sections were washed twice for 5 minutes in Sample Prep Wash Buffer and incubated for 15 minutes on a rocker with either 3 ml of Verification Staining Reagent (ref. 20300014, Vizgen, for verification samples) or 3 ml of DAPI and PolyT Staining Reagent (ref. 20300021, Vizgen, for experimental samples).

Techniques: Labeling, Staining, Expressing, Marker