|
| Status |
Public on Nov 29, 2024 |
| Title |
72-Treated |
| Sample type |
protein |
| |
|
| Source name |
Brain 3D6 treatment
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Brain
|
| Treatment protocol |
PDAPP mice received weekly subcutaneous injections of 3D6 (anti-Aβ1-x,IgG2b) (25mg/kg) or twice-weekly subcutaneous injections of IgG (IgG2a isotype) (50mg/kg) for three months
|
| Growth protocol |
n/a
|
| Extracted molecule |
protein |
| Extraction protocol |
Assay was performed on 5 μm thick FFPE (Formalin-fixed paraffin-embedded) coronal brain sections from IgG- and 3D6-treated PDAPP mice. Sections were mounted onto charged slides and underwent baking at 65°C in a drying oven for 2 hours prior to deparaffinization. Slides were deparaffinized and rehydrated by incubating in CitriSolv (Decon Labs) 3 times for 5min each followed by 2 washes of 100% ethanol for 5 min and 2 washes of 95% Ethanol for 5min and 2 washes of distilled water for 5 min each.
|
| Label |
Unique Multiplex Barcodes
|
| Label protocol |
Slides were removed from the BOND RXm and were incubated overnight at 4°C with a multiplexed cocktail of primary antibodies each of which are conjugated to unique ultraviolent-photocleavable oligonucleotide tags (GeoMx Immune Profile Core and Immune Cell Typing modules containing 31 targets, including three housekeeper and three background probes). Additional fluorescently labeled antibodies were used as morphology markers to select regions of interest (ROIs). In the current study, the morphology markers included β-amyloid (MOAB-2, Novus Biologicals, NBP2-13075AF594), and laminin (Novus Biologicals, NB300-144AF532) to visualize β-amyloid deposition and neuro-vasculature, respectively.
|
| |
|
| Hybridization protocol |
Once all ROIs were processed, pools of released indexing oligos were hybridized to NanoString optical barcodes for digital counting and subsequently analyzed with a nCounter Analysis System. nCounter hybridization assay for photocleaved oligo counting Hybridization of cleaved indexing oligonucleotides to fluorescent barcodes was performed using the nCounter Protein PlexSet reagents based on the manufacturer's directions. Hybridizations were performed at 65 °C overnight in a thermocycler.
|
| Scan protocol |
For each mouse, 12 CAA containing leptomeningeal and 12 CAA containing penetrating vessels were selected as ROIs for high-resolution multiplex profiling, comprising a total of 24 ROIs per animal. A 100-μm in diameter circle was selected as the center ROI surrounding each blood vessel with β-amyloid deposition.
|
| Description |
20221212_P1001660003206E_RCC
1-12 (Leptomeninges) 13-24 (penetrating vessels)
3D6 treated
|
| Data processing |
After hybridization, samples were processed using the nCounter Prep Station and Digital Analyzer as per manufacturer instructions. Data were normalized to technical controls and areas.
Digital counts were normalized with negative controls (Rt IgG2b, Rb IgG, and Rt IgG2a)
|
| |
|
| Submission date |
Jan 16, 2024 |
| Last update date |
Nov 29, 2024 |
| Contact name |
Xavier Taylor |
| Organization name |
Eli Lilly and Company
|
| Street address |
307 E Merrill St
|
| City |
Indianapolis |
| State/province |
Indiana |
| ZIP/Postal code |
46225 |
| Country |
USA |
| |
|
| Platform ID |
GPL34094 |
| Series (1) |
| GSE253388 |
Nanostring protein expression of immune cell recruitment and BBB breakdown following Aβ immunotherapy |
|
