Neonatal Intracerebral Transplantation for Microglia Replacement and Lineage Analysis In Vivo

Summary

This protocol enables efficient neonatal intracerebral transplantation of defined microglia populations, supporting robust long-term engraftment and analysis of microglia lineage and in vivo integration across multiple donor sources and recipient models.

Abstract

Microglia replacement via myeloid cell transplantation has emerged as a promising experimental approach for studying neurological processes and disease. However, existing transplantation strategies are often limited by inefficient engraftment, reliance on irradiation-based conditioning, or restricted flexibility across donor and recipient systems. Here, we describe a neonatal intracerebral transplantation protocol for introducing purified microglia populations (Hoxb8 and non-Hoxb8 microglia) into the developing mouse brain. This method enables efficient donor cell engraftment and integration within the brain parenchyma under physiologically permissive conditions. Importantly, the protocol is compatible with multiple donor sources, including embryonic hematopoietic progenitors and postnatal brain-derived microglia, and can be applied across distinct recipient models with reduced or absent endogenous microglia, including conditional Csf1r-deficient mice and Csf1r^ΔFIRE mice. Donor cells are purified by fluorescence-activated cell sorting and delivered bilaterally into neonatal mouse brains using glass micropipettes for efficient and minimally invasive delivery. Recipient mice are aged to allow donor cells to expand and populate the microglia niche, and outcomes are assessed by histological analysis of donor cell distribution and marker expression. This approach provides a robust and scalable platform for studying microglia ontogeny and in vivo integration across diverse experimental contexts.

Introduction

Microglia replacement through transplantation of myeloid cells has emerged as a powerful strategy for probing microglia biology and as a potential therapeutic paradigm for neurological disease. Over the past several years, the field has advanced to strategies capable of widespread microglia replacement in the central nervous system¹. Recent studies have demonstrated that donor-derived macrophages and progenitors can engraft the brain, adopt microglia-like transcriptional programs, and in some cases improve disease-associated phenotypes^{2,3,4,5,6,7,8}. These findings support the concept that brain macrophage replacement can be leveraged to study and potentially modify neurological disease processes.

A central challenge in microglia transplantation is achieving robust engraftment while minimizing experimental confounds. Many established replacement strategies rely on irradiation, chemotherapy, or microglia depletion paradigms to generate an engraftable niche^{6,9,10,11,12,13}. Despite their effectiveness, these approaches can induce microglia senescence, disrupt the blood-brain barrier, and alter inflammatory signaling, all of which can complicate the interpretation of donor cell behavior^9,14. More recent work has begun to explore conditioning-independent or reduced toxicity approaches to microglia replacement^5,8,15.

Developmental timing is an important determinant of transplantation success. The neonatal brain provides a permissive environment that supports donor cell expansion and long-term integration, likely due to reduced structural barriers and ongoing establishment of the endogenous microglia niche. Recent transplantation studies using early postnatal delivery (P0–P5) demonstrate robust parenchymal engraftment and acquisition of microglia-like identity, reinforcing the importance of developmental context for efficient replacement^7,8,16,17.

Hoxb8 microglia provide a particularly informative system for studying lineage-specific contributions to microglia biology. These cells arise from embryonic hematopoietic progenitors and represent a defined subset of parenchymal microglia with behavioral consequences when dysfunctional^{17,18,19,20,21}. Recent work using primary Hoxb8 progenitors and microglia and estrogen receptor (ER)-Hoxb8 progenitor systems demonstrates that these cells can be expanded, genetically manipulated, and successfully engrafted into the brain, where they adopt microglia-like transcriptional profiles and functional characteristics^7,8,17. These advances further highlight the utility of transplantation approaches for dissecting microglia lineage and heterogeneity.

Here, we describe a neonatal intracerebral transplantation platform optimized for versatility across donor and recipient systems. In contrast to approaches that depend on irradiation or systemic conditioning, this method uses direct bilateral intracerebral delivery into neonatal mice, enabling donor cell placement in a developmentally permissive brain environment while minimizing systemic perturbation. The technique is compatible with multiple donor sources, including embryonic hematopoietic progenitors and postnatal brain-derived microglia, and can be applied across distinct recipient models with reduced or absent endogenous microglia. A major practical advantage is that the thin neonatal skull allows rapid manual injection with glass micropipettes, eliminating the need for drilling or stereotaxic fixation while still achieving broad parenchymal engraftment.

Together, this approach provides a robust, flexible, and scalable system for studying microglia ontogeny, heterogeneity, and in vivo integration across diverse experimental contexts. This positions neonatal intracerebral transplantation as a complementary and experimentally controlled alternative to systemic microglia replacement strategies.

Protocol

All methods and experiments in this study have been performed on mice. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah, Public Health Service Assurance #D16-00018 (A3031-01).

1. Preparing breeding pairs for donor and recipient litters

1. Place breeder pairs in the same cage in the evening to initiate timed matings.
2. Inspect females each morning for the presence of a vaginal plug to confirm successful mating.
3. Remove plugged females from males and house them individually until parturition, which takes approximately 18–20 days.
4. Handle pregnant dams gently for 5 min daily for 7–10 days before birth to reduce stress associated with neonatal handling.
5. Expose dams to a cotton swab containing betadine odor during the daily handling sessions to reduce maternal stress when pups are returned after surgery.
6. Prepare clean cages with nesting material for pregnant females prior to expected delivery.

2. Harvesting and processing neonatal brains

1. Euthanize neonatal donor mice (P0–P4) by isoflurane overdose using a bell jar or similar enclosed chamber equipped with a false floor to prevent direct contact with the anesthetic.
2. Once respiration ceases and the absence of reflexes is confirmed, immediately decapitate.
3. Harvest the whole brain, including the cerebellum and olfactory bulbs. Place brains in 3 mL of cold 1× Hanks’ Balanced Salt Solution (HBSS) in a plastic Petri dish on ice.
4. Using sterile scalpels and forceps, mince the brain into approximately 1–2 mm³ pieces.
5. Transfer the minced brain tissue (1 brain per tissue dissociation tube) into tissue dissociation tubes containing enzymatic digestion reagents supplied with the neural tissue dissociation system according to the manufacturer’s recommended protocol.
6. Place the tubes in a mechanical dissociation device with heating capability and incubate at 37 °C for 30 min using the manufacturer’s recommended neural tissue dissociation program.
7. Pre-soak 70 µm cell strainers with 400 µL of 1× HBSS.
8. Pass the dissociated tissue through the pre-soaked strainers that are placed over a 50 mL conical tube on ice.
9. Wash the tubes with 10 mL of 1× HBSS and pass through filters to ensure maximal cell recovery.
10. Discard filters and spin cells down at 300 × g for 10 min at room temperature. Aspirate the supernatant.
11. Resuspend cells in 1 mL of red blood cell lysis buffer in sterile ultrapure water per sample and incubate for 10 min at 4 °C.
12. Quench the lysis reaction with 10 mL of 1× HBSS/1% BSA/5% FBS.
13. Discard filters and spin cells down at 300 × g for 10 min at 4 °C. Aspirate the supernatant.
14. Determine cell numbers using a hemocytometer. Typical range of cell yield is 3.2–4.0 × 10⁶ prior to sorting.
15. Resuspend cells in 5% FBS in 1× HBSS at the desired concentration for downstream antibody staining.

3. Cell antibody staining for FACS sorting of microglia populations

1. Resuspend cells in 1 mL of staining buffer (5% FBS in 1× HBSS) containing Fc receptor blocking antibody (purified CD16/32, 1:2000) and incubate cells for 10 min at 4 °C.
2. Spin cells down at 240 × g for 3 min at 4 °C. Aspirate the supernatant.
3. Incubate cells with fluorescently conjugated antibodies (CD45 APC, 1:160; CD11b Alex Fluor 700, 1:160) in 1 mL of staining buffer for 30 min on ice in the dark.
4. Spin cells down at 240 × g for 3 min at 4 °C. Aspirate the supernatant.
5. Add a viability dye (e.g., 3 µM DAPI in 1× HBSS) to exclude dead cells.
6. Perform FACS sorting using a high-speed cell sorter equipped with appropriate lasers for DAPI, GFP, tdTomato, CD45-APC, and CD11b-Alexa Fluor 700 detection.
7. Use a 100 µm nozzle with low sheath pressure when possible to maximize cell viability. Keep the sample collection tubes and sorted cells on ice throughout the procedure.
8. Use unstained cells, single-color compensation controls, and fluorescence-minus-one controls when needed to set voltages, compensation, and gates.
9. First gate cells by forward scatter area versus side scatter area to exclude debris.
10. Gate singlets using side scatter height versus side scatter width.
11. Exclude dead cells by gating on DAPI⁻ cells.
12. From live singlets, gate on CD45^low CD11b⁺ microglia.
13. Within the CD45^low CD11b⁺ microglia gate, use GFP and tdTomato fluorescence to separate donor microglia populations (Hoxb8 microglia: DAPI⁻ CD45^low CD11b⁺ tdTomato⁺ GFP⁺, non-Hoxb8 microglia: DAPI⁻ CD45^low CD11b⁺ tdTomato⁻ GFP⁺).
14. Sort each population into sterile collection tubes containing cold 1x HBSS.
15. After sorting, reanalyze a small aliquot of sorted cells to confirm purity and viability before transplantation.
16. Collect sorted cells in sterile 1× HBSS and maintain them on ice in the dark until transplantation.
    NOTE: Typical yields for Hoxb8 microglia range from 3.70–4.60 x 10⁴ cells and non-Hoxb8 microglia 1.10–1.14 x 10⁵ cells per neonatal brain (P0–P4), although yields may vary depending on age, genotype, and tissue processing efficiency. Transplant purified live cells as soon as possible, ideally within 60 min of cell sorting.

4. Harvesting and processing fetal liver hematopoietic progenitors

1. Euthanize pregnant dams using 5% isoflurane delivered in oxygen through a precision vaporizer until respiration ceases and the absence of reflexes is confirmed.
2. Following isoflurane overdose, perform a secondary physical method by cervical dislocation to ensure death, in accordance with institutional animal care and use guidelines.
3. Remove the uterine horns from the pregnant mice and place them in cold 3 mL of 1× HBSS in plastic Petri dishes (60 mm × 15 mm).
4. Dissect E12.5 embryos from the uterus and place in fresh cold 3 mL of 1× HBSS in plastic Petri dishes.
5. Dissect fetal livers using fine forceps and place them in fresh 3 mL of cold 1× HBSS containing 5% FBS in Petri dishes.
6. Mechanically dissociate pooled fetal liver tissue using a 26 G needle and a 3 mL syringe.
7. Spin cells down at 240 × g for 5 min at 4 °C. Aspirate the supernatant.
8. Wash cells with 1× HBSS.
9. Pass the cell suspension through an 80 µm cell strainer.
10. Incubate cells in 20 µL of red blood cell lysis buffer (0.15 M ammonium chloride, 1 mM KHCO3) for 5 min followed by adding 13 mL of 1× HBSS for quenching.
11. Continue incubation for 5 min to lyse red blood cells.
12. Spin cells down at 200 × g for 5 min at 4 °C.

5. Cell antibody staining for FACS sorting of hematopoietic progenitor cells

1. Incubate fetal liver cells with fluorescently conjugated antibodies (Ter119 PerCP-Cy5.5, 1:50; Kit PE-Cy7, 1:100) in 5% FBS/1% BSA in 1× HBSS for 30 min on ice in the dark.
2. Wash cells with 1× HBSS and spin down at 240 × g for 5 min at 4 °C.
3. Add a viability dye (e.g., 3 µM DAPI in 1× HBSS) to exclude dead cells.
4. Perform FACS sorting using a high-speed cell sorter equipped with appropriate lasers and filters for DAPI, Ter119 PerCP-Cy5.5, Kit PE-Cy7, tdTomato, and GFP detection.
5. Use a 100 µm nozzle with low sheath pressure when possible to maximize cell viability. Keep the sample collection tubes and sorted cells on ice throughout the procedure.
6. Use unstained cells, single-color compensation controls, and fluorescence-minus-one controls when needed to set voltages, compensation, and gates.
7. First gate cells using forward scatter area versus side scatter area to exclude debris.
8. Gate singlets using forward scatter height versus forward scatter width, followed by side scatter height versus side scatter width.
9. Exclude dead cells by gating on DAPI⁻ cells.
10. From live singlets, exclude erythroid lineage cells by gating on Ter119⁻ cells.
11. Within the Ter119⁻ population, gate on Kit^high hematopoietic progenitor cells.
12. Within the DAPI⁻ Ter119⁻ Kit^high population, isolate Hoxb8 lineage-positive progenitors by gating on tdTomato⁺ cells.
13. If the donor line also carries the Cx3cr1-GFP reporter, use GFP fluorescence to confirm that the sorted fetal liver progenitor population is GFP⁻ or GFP^low at this stage, distinguishing progenitor cells from differentiated Cx3cr1-expressing myeloid cells.
14. Sort the final DAPI⁻ Ter119⁻ Kit^high tdTomato⁺ GFP^- population into sterile collection tubes containing cold 1× HBSS.
15. After sorting, reanalyze a small aliquot of sorted cells to confirm purity and viability before transplantation.
16. Collect sorted cells in sterile 1× HBSS and maintain them on ice in the dark until transplantation.
    NOTE: Typical yields range from approximately 5.0–7.0 × 10⁴ Hoxb8 hematopoietic progenitors per E12.5 fetal liver preparation, depending on embryo size and sorting efficiency. Transplant purified live cells as soon as possible, ideally within 60 min of sorting.

6. Preparing injection needles

1. Load borosilicate glass capillaries (1.0 mm outer diameter, 0.58 mm inner diameter, with filament) into a micropipette puller.
2. Pull capillaries using a two-line pulling program optimized to generate long tapered needles suitable for neonatal intracerebral transplantation.
   1. Typical settings include: Heat = 645, Pull = 30, Velocity = 120, Delay = OFF and Pressure = 200. Optimize the final settings for the specific glass type, environmental conditions, and puller calibration. Subsequently, trim the resulting needles and bevel them to produce a final outer tip diameter of approximately 30 µm.
3. Place the pulled needles onto two strips of putty inside a large plastic Petri dish to protect the tips from damage before trimming and beveling.
4. Place the pulled needle under a dissecting stereo microscope at approximately 10×–20× magnification and support it with a soft brace.
   NOTE: Higher magnification (20×–40×) may be used during tip inspection to verify proper taper formation and identify any defects prior to trimming and beveling.
5. Use a sterile razor blade to trim the needle tip to obtain an outer diameter of approximately 30 µm.
6. Secure the trimmed needle in a microgrinder holder.
7. Adjust the bevel angle to 30°–35°.
8. Turn on the grinder and illumination.
9. Allow distilled water to drip onto the grinding stone at a steady rate of approximately one drop every 4–5 s.
10. Bring the needle tip gently into contact with the rotating grinding stone until a beveled edge is formed.
11. Measure the outer diameter of the beveled tip using a tip measuring device when available.
12. Select needles with an outer diameter close to 30 µm for transplantation.
13. Fill the needle with sterile buffer and gently expel liquid to confirm that the tip is open and unobstructed.

7. Preparing the injection apparatus

1. Assemble a manual mouth-controlled pipette consisting of flexible tubing, a mouthpiece, an inline filter, and a capillary holder.
   NOTE: An inline filter is incorporated into the mouth-controlled pipetting apparatus to prevent contamination.
2. Mark the needle every 2 mm (up to 2 marks per needle). Every 2 mm corresponds to ~2 µL.
3. Insert the prepared glass injection needle ~1.5 cm into the capillary holder and confirm that the assembly is secure and airtight.
4. Apply gentle suction through the mouthpiece to verify unobstructed airflow and proper resistance.
5. Confirm that the system allows smooth control of aspiration and injection before loading donor cells.
6. Backfill the glass injection needle with the prepared donor cell suspension (4.5 µL total) using a fine pipette tip.
   NOTE: Ensure that no air bubbles remain in the needle before beginning transplantation. Use one needle per mouse.

8. Anesthetizing neonatal recipients and preparing the surgical field

1. Position the neonatal mouse in the supine orientation on the surgical table and induce anesthesia using 2.0%–5.0% isoflurane delivered in oxygen through a precision vaporizer.
2. Place the mouse nose within a nose cone to maintain continuous delivery of isoflurane and oxygen throughout the procedure.
3. Allow approximately 1 min for anesthesia induction while monitoring respiration.
4. Gently touch the limbs and confirm the absence of a withdrawal reflex to verify adequate anesthesia.
5. Once an appropriate anesthetic plane is achieved, reduce the isoflurane concentration to 0.5%–2.0% for maintenance and continue anesthesia for up to 10 min during the transplantation procedure.
6. Maintain the neonatal mouse at approximately 38 °C using a heat pad placed underneath the surgical table.
7. Clean the injection site area on the head using alternating applications of betadine and 70% ethanol with sterile cotton swabs.
8. Apply a final layer of betadine and allow the skin to dry before injection.
   CAUTION: Isoflurane is a hazardous anesthetic agent. Use appropriate anesthetic scavenging systems and ensure adequate room ventilation, in accordance with institutional safety guidelines.

9. Performing neonatal intracerebral transplantation

1. Gently stabilize the head using gloved fingers or a soft support to minimize movement during injection.
   NOTE: The recipient mice are transplanted between P1 and P4.
2. Identify bilateral injection sites in the anterior dorsal telencephalon (future frontal cortex region), approximately 1–2 mm lateral to the sagittal midline and immediately caudal to the olfactory bulbs. Position the injection sites symmetrically in each hemisphere while avoiding visible surface blood vessels.
   NOTE: Due to the thin skull and small size of neonatal mice, injections are performed using visual anatomical landmarks rather than stereotaxic coordinates.
3. Visually inspect the skull and avoid inserting the needle near visible blood vessels.
4. Advance the beveled glass micropipette gently through the skin and developing skull.
5. Guide the needle approximately 1–2 mm below the skull surface into the frontal hemisphere.
6. Deliver donor cells using controlled manual pressure through the mouth-controlled pipette apparatus.
   NOTE: When transplanting purified hematopoietic progenitors or neonatal microglia, inject 2–3 µL per hemisphere, corresponding to approximately 2.5 × 10⁴ cells per injection. This provides approximately 5 × 10⁴ cells per mouse brain.
7. Slowly withdraw the micropipette from the brain to minimize reflux of injected cells and reduce tissue damage.
8. Perform contralateral injection.
   NOTE: Use one needle per mouse to avoid cross-contamination between mice.

10. Recovering transplanted neonates

1. Place each pup on a warming pad immediately after the procedure.
2. Monitor the pups until normal body color, spontaneous movement, and overall activity return.
   NOTE: Recovery time ranges from 5 min to 10 min.
3. Return pups to the dam.
4. Rub each pup thoroughly with home-cage bedding and nesting material.
5. Place pups back in the cage with the dam.
6. Confirm that the dam retrieves and accepts the returned pups.

11. Monitoring postoperative health

1. Observe pups daily for 10 min for the first 3 days after transplantation.
2. Evaluate pups for normal activity, suckling behavior, and interaction with the dam.
   NOTE: Do not disturb mouse cages during this time. This can increase stress on the dam.
3. Continue monitoring pups every other day until weaning after the initial 3-day period.
4. Evaluate pups for normal development, feeding behavior, activity level, and maternal care.
5. Measure and record body weight daily during the early postoperative period.
6. Assess pups for signs of distress, abnormal posture, lethargy, or impaired feeding.
7. Perform humane euthanasia when necessary according to institutional animal care guidelines.
   1. For neonatal mice, euthanize by isoflurane overdose using a bell jar or similar enclosed chamber equipped with a false floor to prevent direct contact with the anesthetic. Once respiration ceases and the absence of reflexes is confirmed, immediately decapitate.
   2. For adult mice, euthanize using 5% isoflurane delivered in oxygen through a precision vaporizer until respiration ceases and the absence of reflexes is confirmed, followed by cervical dislocation as the secondary physical method to ensure death.
      NOTE: Animals exhibiting postoperative mortality or absence of detectable donor cell engraftment at the experimental endpoint were excluded from analysis. Postoperative analgesia was not administered because neonatal intracerebral transplantation was considered minimally invasive.

12. Collecting brain tissues for cryosectioning and immunofluorescence staining

1. Remove the brain carefully and prepare it for frozen-section analysis.
2. Process postnatal brain tissue for cryosectioning and cut coronal sections using a cryostat according to previously described procedures.
   NOTE: For each mouse, 9–12 coronal sections spanning the somatosensory cortex are analyzed.
3. Mount sections onto microscope slides and store them at -20 °C until staining.
4. Incubate sections briefly in permeabilization buffer containing 0.2% Triton X-100 and 1% sodium deoxycholate.
5. Prepare primary antibodies (chicken anti-GFP, 1:500; Guinea pig anti-tdTomato antibody, 1:250; rabbit anti-Iba1; 1:500) diluted in appropriate antibody dilution buffer.
6. Incubate tissue sections with primary antibodies overnight at 4 °C.
7. Wash sections with phosphate-buffered saline to remove unbound primary antibodies.
8. Incubate sections with fluorophore-conjugated secondary antibodies (goat anti-rabbit Alexa Fluor 647, 1:500; goat anti-guinea pig Alexa Fluor 555, 1:500; goat anti-chicken Alexa Fluor 488, 1:500) for 2 h at room temperature in the dark.
9. Counterstain sections with DAPI to visualize cell nuclei.
10. Mount sections using an antifade mounting medium and apply a glass coverslip.

13. Acquiring confocal microscopy images

1. Acquire fluorescence images using a confocal microscope equipped with 10× or 20× objectives.
2. Acquire images at 512 × 512 or 1024 × 1024 pixel resolution using scan speeds of 200–400 Hz.
3. Collect optical sections with 2–5 µm z-step intervals through the tissue.

Results

Neonatal intracerebral transplantation of purified microglia populations results in long-term survival and widespread engraftment of donor-derived cells within the recipient brain parenchyma. Purified donor microglia populations were isolated by fluorescence-activated cell sorting using fluorescent lineage reporters (Hoxb8 microglia: tdTomato⁺ GFP⁺; non-Hoxb8 microglia: tdTomato⁻ GFP⁺) prior to transplantation (Figure 1A). Following neonatal intracerebral transplantation, d...

Discussion

Neonatal intracerebral transplantation of purified microglial progenitors or defined microglial subpopulations provides a robust approach for reconstructing the microglial compartment in vivo and examining lineage-specific properties of brain macrophages. This method enables selective reconstitution using genetically defined donor cells, allowing direct assessment of donor cell engraftment and maturation within the brain environment.

A key advantage of this strategy is its compatibili...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bell jar	Various	N/A	Used for euthanasia
Borosilicate glass capillaries with filament	Sutter Instrument	BF100-78-15	Used for injection needles
Bovine serum albumin (BSA)	Sigma Aldrich	A8806	Used to prepare PB buffer. Various brands are available. The one listed here is just an example.
CD11b Alexa Fluor 700	BioLegend	101222	Myeloid cells
CD45 APC	BioLegend	103112	Hematopoietic cells
Cell strainers (70 µm)	Falcon	352350	Used to generate single- cell suspensions. Various brands are available. The one listed here is just an example.
Chicken anti-GFP antibody	Aves Labs	GFP-1020	Immunofluorescence staining
c-Kit PE-Cy7 antibody	BioLegend	105814	Hematopoietic progenitors
Confocal microscope	Leica	TCS SP5	Used for fluorescence imaging
Conical tubes (15 mL/ 50 mL)	Eppendorf	https://www.eppendorf.com/us-en/Products/Lab-Consumables/Lab-Tubes/Eppendorf-Tubes-BioBased-p-PF-4440301	Tissue/cell processing. Various brands are available. The one listed here is just an example.
Cryostat machine	Leica	https://www.leicabiosystems.com/en-de/histology-equipment/cryostats/	Generate frozen tissue sections. Various brands are available. The one listed here is just an example.
DAPI nuclear stain	Thermo Fisher Scientific	D1306	Used for viability and nuclear staining
Dissecting microscope	Leica	https://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/	Used for trimming and preparing injection needles. Various brands are available. The one listed here is just an example.
FACSAria Flow Cytometer Sorter	BD Bioscience	FACSAria	Cell purification
Fc block (purified CD16/32)	Biolegend	101302	Blocks unwanted antibody binding to mouse cells expressing Fc receptors
Fetal bovine serum (FBS)	ThermoFisher Scientific	https://www.thermofisher.com/in/en/home/life-science/cell-culture/mammalian-cell-culture/fbs.html	Used in cell preparation buffers. Various brands are available. The one listed here is just an example.
Flexible tubing for mouth pipette	Various	N/A	Component of manual injection apparatus
Flow cytometry analysis software	FlowJo	v10.8.1	Used for FACS data analysis
Flow cytometry cell sorter	BD Biosciences	FACSAria	Used for cell purification
gentleMACS Octo Dissociator with heaters	Miltenyi Biotec	130-096-427	Used for brain tissue dissociation
Goat anti-chicken Alexa Fluor 488 secondary antibody	Thermo Fisher Scientific	A-11039	Secondary antibody
Goat anti-guinea pig Alexa Fluor 555 secondary antibody	Thermo Fisher Scientific	A-21428	Secondary antibody
Goat anti-rabbit Alexa Fluor 647 secondary antibody	Thermo Fisher Scientific	A-21245	Secondary antibody
Goat anti-rat Alexa Fluor 647 secondary antibody	Thermo Fisher Scientific	A-48265	Secondary antibody
Guinea pig anti-tdTomato antibody	Frontier Institute	AB_2631185	Immunofluorescence staining
Hanks’ balanced salt solution (HBSS)	Gibco	14175-079	Used during cell preparation
Hemocytometer	Various	N/A	Cell counting
Inline filter for mouth pipette	Various	N/A	Prevents contamination
Microgrinder	Narishige	EG-400	Used to bevel glass injection needles
Micropipette puller	Sutter Instrument	P-1000	Used to generate fine injection needles
Mouth pipette mouthpiece	Various	N/A	Used for manual control of injection
Neural Tissue Dissociation Kit (P)	Miltenyi Biotec	130-092-628	Used for enzymatic dissociation of brain tissue
Petri dishes	Thermo Fisher Scientific	https://www.thermofisher.com/search/browse/category/us/en/90111022	Used for storing pulled needles or tissue harvest. Various brands are available. The one listed here is just an example.
Rabbit anti-Iba1 antibody	Wako	019-19741	Microglia marker
Rabbit anti-TMEM119 antibody	Abcam	209064	Microglia-specific marker
Rat anti-CD206 antibody	BioLegend	141712	Used to identify macrophage populations
Rat anti-P2RY12 antibody	BioLegend	848002	Microglia marker
Razor blades/scalpels	Various	N/A	Used to trim needle tips or tissue harvest
RBC lysis buffer	ChemCruz	sc-296258	Used for removal of red blood cells
Stereotaxic apparatus	Kopf Instruments	https://kopfinstruments.com/	Used for surgical implantation. Various brands are available. The one listed here is just an example.
Ter119 PerCP-Cy5.5 antibody	BioLegend	116228	Erythroid lineage cells