Aug 27, 2025
Version 1
Carver et al, Aged Brain Spatial Profiling - Tissue Processing V.1
- Chase Carver1
- 1Mayo Clinic
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Chase Carver 2025. Carver et al, Aged Brain Spatial Profiling - Tissue Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x841g25/v2Version created by Chase Carver
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 04, 2023
Last Modified: August 27, 2025
Protocol Integer ID: 88812
Keywords: modifiable features of aged brain white matter, aged brain white matter, aged brain spatial profiling, steps for mouse brain tissue processing, mouse brain tissue processing, associated microglia, tissue processing, mouse brain, image brain sections in the respective technique, tissue processing this protocol, image brain section, immunohistochemistry
Funders Acknowledgements:
NIH Cellular Senescence Network
Grant ID: UG3 CA275669-01
Abstract
This protocol provides the steps for mouse brain tissue processing and preparation for use in immunohistochemistry and spatial -omic techniques used in Carver et al., "Senescent- and disease-associated microglia are modifiable features of aged brain white matter"
The steps here should be considered as immediately proceeding the protocol for each separate process to correctly stain and image brain sections in the respective techniques.
Troubleshooting
Perfusion
Euthanize mouse with intraperitoneal injection of 32.5mg/ml pentobarbital
Extract blood via inferior vena cava with a syringe
Perfuse transcardially with ice-cold PBS
Brain Processing
Decapitate mouse and extract brain from skull
Bisect the two hemispheres of the brain
Submerge right hemisphere in 4% PFA in PBS for 24 hours
Microdisect left hemisphere hippocampus from hippocampus adjacent white matter, place each tissue in 1 mL TRIzol in ceramic bead tube for downstream RNA processing
Brain sections for immunohistochemistry
Steps for cutting brain slices for traditional immunofluorescence
In sucrose solutions, brain should initially float and then eventually sink over the time period in each step.
Transfer brain from PFA to 10% sucrose in PBS for 24 hours
Transfer brain from 10% sucrose to 20% sucrose in PBS for 24 hours
Transfer brain from 20% sucrose to 30% sucrose in PBS for 24 hours
Cryopreserve brain hemisphere in 30% glycerol, 30% ethylene glycol, 40% PBS w/v at -80C
Embed brain in Tissue-tek O.C.T. compound and freeze the brain block in a cassette on top of dry ice
Cut sagittal sections at 30 μm thickness on a Leica CM3050 S cryostat at -20C
Place sections in a well plate containing ice-cold PBS + 0.01% sodium azide and maintain plate in 4C environment prior to further processing
Brain sections for spatial -omics
Steps for cutting brain slices for spatial -omics profiling for GeoMx and CosMx
Transfer PFA-fixed brain to cold 70% ethanol
Embed brain in paraffin wax block within 24 hours
Cut sagittal sections at 5 μm thickness with a sliding microtome
Place cut paraffin section in deionized water bath heated to 40-42C
Mount the section on a Superfrost Plus glass slide by placing the slide submerged under the section and pulling upward such that the section is within the correct window for GeoMx or CosMx (see attachment)
Allow water to drain from slide and dry and bake slide for 30 min at 60C