hybridization 667 buffer  (Molecular Instruments)

Journal: bioRxiv

Article Title: Harnessing TfR1 for Cross-Species Systemic Delivery of siRNAs to Deep Brain Regions Using Single-Domain Antibodies

doi: 10.64898/2026.05.20.726486

Figure Lengend Snippet: (A, B) The C5-hFc-siSOD1 conjugate was dosed in WT mice as a single IV or SC bolus at 1.5 mg/kg (A) or 0.14 to 9 mg/kg siRNA equivalent dose (B). Target knockdown was measured 14 days after administration, using RT-qPCR on total RNA extracts from indicated regions of the CNS. The results are expressed as a ratio to mRNA levels measured in PBS-treated animals after normalization to RpL13 and RpL30 reference genes. (A) Box plots: extends from the 25th to 75th percentiles with center line plotted at the median. Whiskers indicate the smallest and the largest experimental values (n = 8-16 animals per group from 3 independent experiments). (B) Dose-response curves in each CNS region, where experimental results are expressed as means ± SEM, and estimated ED 50 values. (C) Mice were administered with either multi-IV (Q2Dx3) at 1.5 mg/kg or single SC at 4.5 mg/kg siRNA equivalent dose of the C5-hFc-siSOD1 conjugate. Shown are in situ hybridization micrographs showing siSOD1 AS distribution (top panels, miRNAScope®) throughout the brain (sagittal sections) and spinal cord (cross-sections), and corresponding Sod1 mRNA (lower panels, RNAScope®), 14 days after the last IV or single SC (representative animal from n=4 mice per group). (D) Magnifications and neuron-specific co-staining, using a Map2 mRNA-specific probe, in indicated regions of the brain.

Article Snippet: For knock-down analysis on tissue sections using in situ hybridization (RNAScope, ACD), left cerebral hemisphere half-slices were cut into one ventral and one dorsal parts and immediately immersed in 10% neutral buffer formalin (NBF) fixative solution for approximately 48h at RT.

Techniques: Knockdown, Quantitative RT-PCR, In Situ Hybridization, RNAscope, Staining

Journal: bioRxiv

Article Title: Harnessing TfR1 for Cross-Species Systemic Delivery of siRNAs to Deep Brain Regions Using Single-Domain Antibodies

doi: 10.64898/2026.05.20.726486

Figure Lengend Snippet: (A-B) In situ hybridization micrographs showing siSOD1 AS distribution (miRNAScope®) throughout the brain (sagittal sections) and spinal cord (cross-sections) (A), 14 days after multi-IV bolus (Q2D x3) of the non-binding C5neg- or the TfR1-binding B8V32-hFc-siSOD1 conjugates, at 1.5 mg/kg siRNA. (B) In situ hybridization micrographs showing siSOD1 AS distribution (top panels, miRNAScope®) and corresponding Sod1 mRNA (lower panels, RNAScope®) in retina layers of the same animals (representative animal from n=4 mice per group). Retina layers: GCL, Ganglion Cell Layer; IPL, Inner Plexiform Layer; INL, Inner Nuclear Layer; OPL, Outer Plexiform Layer; ONL, Outer Nuclear Layer; IS, Inner Segments; OS, Outer Segments; RPE, Retinal Pigment Epithelium; Chor, Choroid.

Article Snippet: For knock-down analysis on tissue sections using in situ hybridization (RNAScope, ACD), left cerebral hemisphere half-slices were cut into one ventral and one dorsal parts and immediately immersed in 10% neutral buffer formalin (NBF) fixative solution for approximately 48h at RT.

Techniques: In Situ Hybridization, Binding Assay, RNAscope

Journal: bioRxiv

Article Title: Harnessing TfR1 for Cross-Species Systemic Delivery of siRNAs to Deep Brain Regions Using Single-Domain Antibodies

doi: 10.64898/2026.05.20.726486

Figure Lengend Snippet: Cynomolgus monkeys were dosed by 30-min IV infusion three times at 7 days intervals with the B8V32-hFc-siHPRT conjugate, at the same dose previously evaluated in hTfR-KI mice of 1.5 mg/kg siRNA equivalent dose. Plasma samples were collected at indicated timepoints for PK analysis (A). Tissues were recovered at 14 days after last injection to assess target knockdown using RT-qPCR (B) and in situ hybridization in the brain using RNAScope® on one coronal section through the putamen (top panels: approximately -1 to -3 mm from the anterior commissure, based on Calabrese et al. 2015) and one through the hippocampus (lower panels: approximately -11 to -13 mm from the anterior commissure; star indicates an artefact) (C). (D) Neuroinflammation was assessed on brain tissue samples by measurement of mRNA levels of IbA1, GFAP and C3 as markers of microglial reactivity, astrocyte reactivity and complement activation, respectively.

Article Snippet: For knock-down analysis on tissue sections using in situ hybridization (RNAScope, ACD), left cerebral hemisphere half-slices were cut into one ventral and one dorsal parts and immediately immersed in 10% neutral buffer formalin (NBF) fixative solution for approximately 48h at RT.

Techniques: Clinical Proteomics, Injection, Knockdown, Quantitative RT-PCR, In Situ Hybridization, RNAscope, Activation Assay