Journal: bioRxiv

Article Title: Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus

doi: 10.1101/2024.11.07.622523

Figure Lengend Snippet: a Brightfield image of FFPE human cerebellum following barcoding in the DBiTplus workflow. Box represents the barcoded region. Brightfield image after cDNA retrieval shows the tissue morphology is preserved and is suitable for CODEX imaging. b The same tissue section from panel A is imaged with a 28-marker panel. Left: CODEX image showing AQP4 (Astrocytes), MAP2 (Synaptic neurons), NeuN (Neuronal nuclei and cell bodies) and Vimentin (Vasculature). Right top: Synaptic neurons marked by MAP2 in the molecular layer of the cerebellum. Right bottom: Large pear-shaped Purkinje cells, with their large cell bodies visualized in the Purkinje cell layer. c Brightfield image of FFPE of benign human lymph node. Box represents the barcoded region. Brightfield image after cDNA retrieval shows the tissue morphology is preserved and is suitable for CODEX imaging. d Size distribution from TapeStation traces of cDNA amplicon. e CODEX imaging on FFPE of human lymph node following the DBiTplus workflow. Three distinct regions are further analyzed for quality of CODEX staining. f Top: Region showing T cell subtypes: CD3ɛ (T cells), CD8 (Cytotoxic T cells), CD4 (Helper T cells) and SMA (Smooth muscles). Middle: CD3ɛ (T cells), SMA (Smooth muscles), CD20 (B cells) and CD21 (Follicular dendritic cells). Bottom: SMA (Smooth muscles), Ki67 (Proliferating germinal center B cells), Collagen IV (Blood vessels), CD31 (Vasculature).

Article Snippet: Human brain cerebellum paraffin sections ( HP-202 ), were purchased from Zyagen and made of freshly harvested tissues, fixed in 10% neutral buffered formalin, and processed for paraffin embedding.

Techniques: Imaging, Marker, Amplification, Staining, Muscles

Journal: Journal of Human Genetics

Article Title: A homozygous nonsense variant in the alternatively spliced VLDLR exon 4 causes a neurodevelopmental disorder without features of VLDLR cerebellar hypoplasia

doi: 10.1038/s10038-024-01279-w

Figure Lengend Snippet: Qualitative and quantitative VLDLR transcript analysis in patient-derived fibroblasts. A Sequence traces showing VLDLR mutant mRNAs with the nonsense variant c.376C > T in fibroblasts from patients 1 and 2. An arrow points to the nucleotide change. B Agarose gel showing RT-PCR amplicons of VLDLR transcripts with primers located in exons 3 and 5 using cDNA derived from patient- and control-fibroblasts, human cerebellum, and fetal brain. The lower band of 272 bp represents VLDLR transcripts without exon 4 and the upper band (395 bp) transcripts with exon 4. C Sequence traces of colony PCR products from cloned RT-PCR amplicons of patient 1. The upper sequence shows VLDLR transcripts with exon 4 and the lower sequence VLDLR mRNAs without exon 4. Quantification of total VLDLR mRNA levels with two primer combinations (exons 10-11 and 12-13) ( D ) and of VLDLR exon 4-containing mRNA levels with primers in exons 3 and 4 ( E ) by RT-qPCR. GAPDH mRNA was used as an internal control; the amount of target mRNA relative to GAPDH mRNA is presented. The mean ± SD of four independent experiments, each performed in triplicate, is shown. One-way ANOVA with Dunnett’s correction was used for statistical analysis: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; Ctrl control, Ex exon, ns not significant, P patient

Article Snippet: Total RNA from human brain cerebellum (Zyagen, #HR-202) and human fetal brain (BioChain®, #R1244035-50) were purchased.

Techniques: Derivative Assay, Sequencing, Mutagenesis, Variant Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Control, Clone Assay, Quantitative RT-PCR