CL-NC

Guide
(The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO/PBS buffer.
1.2 Concentration recommendation: 1 mM.

2. Working Solution Preparation
2.1 Diluent: PBS buffer with DMSO.
2.2 Working concentration: 50 µM; 100 µM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type:[1]
3.1.1 Adherent cells: HT22, Bend.3, HEK293, AML-12, H9C2, HeLa cells.
3.1.2 In vivo samples: Murine intracerebral and intraspinal tissues.
3.2 Incubation conditions:
3.2.1 Incubate adherent cells with Tz-IFDL (HY-D3369) (50 µM) at 37°C for 30 min.
3.2.2 After washing, incubate adherent cells with CL-NC (50 µM) at 37°C for 3 h.
3.2.3 For in vivo imaging, administer Tz-IFDL via intracranial/intrathecal injection 1 h prior to CL-NC injection.
3.3 Washing steps:
3.3.1 Wash adherent cells with PBS buffer after Tz-IFDL incubation.

4. Controls
4.1 Negative controls:
4.1.1 Adherent cells: NR2B-knockdown cells, NR2B-null cell lines, cells treated with Pre-NC instead of CL-NC, cells treated with Tz-IFDL plus competitive Ifenprodil (HY-12882).
4.1.2 In vivo: Mice treated with tetrazine-free compound followed by CL-NC; mice treated with Tz-Br followed by CL-NC; mice pre-injected with Ifenprodil before probe administration.
4.2 Positive control validates experimental system; blank control excludes reagent fluorescence interference.

5. Detection & Analysis
5.1 Instrument: Chemiluminescence imaging system
5.1.1 Emission wavelength: 710 nm.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: OFF-ON chemiluminescence signal triggered by bioorthogonal reaction between CL-NC and tetrazine-conjugated ligand; peak intensity at ~3.5 h for CL-NC/Tz-Br system, ~3 h for in vivo imaging; half-life of 13.4 h for CL-NC/Tz-Br system, 8 h for CL-NC/Tz-IFDL system.
5.2.2 Fluorescence localization: Intracerebral and intraspinal regions in mice; cell surface of NR2B-expressing cells.
5.2.3 Color changes: Deep-red/near-infrared chemiluminescence.
5.2.4 The chemiluminescence intensity of wild-type mice was higher than that of Alzheimer's disease model mice (reflecting a decrease in NMDAR expression levels); higher intensity is observed in NR2B-expressing cells compared to NR2B-null/knockdown cells.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.