All antibodies used for immunohistochemistry, FACS analysis, and cell purification are listed in Table S1 , along with vendors, catalog numbers, and dilution factors. Mouse brains were fixed in 4% paraformaldehyde (PFA) in PBS, followed by cryoprotection in 30% sucrose in PBS prior to cryosectioning and histological analyses. Brain sections were permeabilized by 0.3% Triton X-100 in 10% BSA. Sections were incubated with primary antibodies overnight, followed by secondary antibodies. This is the general procedure unless otherwise specifically described. All images were acquired using a Zeiss LSM 880 confocal microscope with Airyscan and processed with Zen black 2.1 or Zen blue lite 2.3 (Carl Zeiss). Detailed methodologies for image acquisition and quantification of cell numbers, synapses, and phagocytosis are described in Supplemental Experimental Procedures.
Zen blue lite 2
Manufactured by Zeiss
Sourced in Germany
Zen blue lite 2.3 is a software package designed for operating and controlling microscopy systems. It provides a user-friendly interface for configuring and managing microscope hardware, as well as capturing and processing images.
Automatically generated - may contain errors
4 protocols using zen blue lite 2
1
Immunohistochemistry and Microscopy Protocols
2
Immunohistochemistry and Microscopy Protocols
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All antibodies used for immunohistochemistry, FACS analysis, and cell purification are listed in Table S1 , along with vendors, catalog numbers, and dilution factors. Mouse brains were fixed in 4% paraformaldehyde (PFA) in PBS, followed by cryoprotection in 30% sucrose in PBS prior to cryosectioning and histological analyses. Brain sections were permeabilized by 0.3% Triton X-100 in 10% BSA. Sections were incubated with primary antibodies overnight, followed by secondary antibodies. This is the general procedure unless otherwise specifically described. All images were acquired using a Zeiss LSM 880 confocal microscope with Airyscan and processed with Zen black 2.1 or Zen blue lite 2.3 (Carl Zeiss). Detailed methodologies for image acquisition and quantification of cell numbers, synapses, and phagocytosis are described in Supplemental Experimental Procedures.
3
miRNA FISH in Hypoxia-Treated C166 Cells
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For miRNA FISH, C166 cells were seeded on 8-well chamber slides and grown in normoxia or hypoxia (24 h). ViewRNA miRNA ISH Cell Assay Kit (Thermo Fisher Scientific) was used for the hybridizations according to the manufacturer’s protocol. ViewRNA Cell Plus Probe Set (Thermo Fisher Scientific) was used to detect miR-210-3p (assay ID: VM1-10263-VCP, detection label Alexa Fluor 546). Cell nuclei were visualized using DAPI stain. Pictures were taken using ZEISS LSM700 confocal microscope using 40× oil objective and analyzed with ZEN lite blue 2.6 software (ZEISS).
4
miRNA FISH in C166 cells
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For miRNA FISH, C166 cells were seeded on 8-well chamber slides and grown in normoxia or hypoxia (24h). ViewRNA miRNA ISH Cell Assay Kit (Thermo Fisher Scientific) was used for the hybridizations according to the manufacturer’s protocol. ViewRNA Cell Plus Probe Set (Affymetrix/Thermo Fisher Scientific) was used to detect mmu-miR-466c-3p (miR-466p-3p, assay ID: VM1-21572, detection label Alexa Fluor 546). Cell nuclei were visualized using DAPI stain. Pictures were taken using ZEISS LSM700 confocal microscope using 40× oil objective and analyzed with ZEN lite blue 2.6 software (ZEISS, Germany).
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