DPBS/BSA buffer: Prepare a solution containing Dulbecco’s phosphate-buffered saline (DPBS) with Ca²⁺ and Mg²⁺ and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with DPBS. Keep buffer cold (2–8 °C). Degas buffer before use, as air bubbles could block the column.
▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).

Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

Use the Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.

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Neural Tissue Dissociation Kits

Isolate CD171 (L1CAM)-positive neurons from the single-cell suspension using the CD171 (L1CAM) MicroBead Kit. Follow the protocol of the kit data sheet.

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CD171 (L1CAM) MicroBead Kit, mouse

To stain and analyze the isolated neurons by flow cytometry, follow the protocol of the data sheet for the Labeling Check Reagent-APC.
▲ Note: Use DPBS/BSA buffer instead of the buffer described in the data sheet.

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Labeling Check Reagent-APC

1. Plate 5×10⁴ cells in 50 μL of pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of cell isolation and cell culture").
2. Let the cells settle down for 30 minutes at 37 °C in the incubator.
3. Carefully add 450 μL of prepared medium to each well.
4. Take off the medium to remove non-attached and dead cells.
5. Add 500 µl of prepared medium.
6. Maintain the culture by replacing 50% of medium every other day.
   

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse (1:10).

1. Wash cells 3× with PBS.
2. Fix cells with 2% PFA for 10 minutes at room temperature.
3. Wash cells 3× with PBS.
   ▲Note: When working with CD68 antibodies, add 0.2% TRITON X-100 in PBS, incubate for 10 minutes at room temperature, and wash cells 3× with autoMACS® Running Buffer. Do not treat cells with TRITON X-100 before staining with Anti-ACSA-2, Anti-O4, or Anti-PSA-NCAM antibodies.
   ▲Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
4. Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
   
5. Discard staining buffer.
6. Add pure antibody of choice in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
   
7. Wash cells 3× with autoMACS Running Buffer.
   
8. Add a corresponding secondary antibody in staining buffer to the cells and incubate in the dark. For incubation temperature and time as well as recommended antibody concentration, refer to the table below.
   
9. Wash cells 3× with autoMACS Running Buffer.
   ▲Note: For co-staining with additional antibodies repeat step 6–9.
10. Store cells in autoMACS Running Buffer.
11. Cells are now ready for immunofluorescence microscopy.
    ▲Note: Samples can be stored at 2–8 °C in the dark for up to one week.
    ▲Note: When working with cells cultured on coverslips, the coverslips need to be mounted onto slides before imaging.
    

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