Tz-IFDL

Operating Instructions
Operating Instructions (The following is a recommended experimental protocol for reference only, and adjustments are required based on specific needs)

1. Stock Solution Preparation
1.1 Solvent: PBS containing 10% DMSO.
1.2 Recommended concentration: 1 mM.

2. Working Solution Preparation
2.1 Diluent: PBS containing 10% DMSO.
2.2 Working concentration: 100 µM for in vivo imaging.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh immediately before use.

3. Staining Procedures
3.1 Sample Types^[1]:
3.1.1 Tissue samples: Mouse brain and spinal cord tissues.
3.1.2 Animal samples: 6-month-old C57BL/6J wild-type mice and APP/PS1 transgenic mice.
3.2 Incubation Conditions:
3.2.1 In vivo brain imaging: Administer 10 µL of 100 µM Tz-IFDL via intracranial injection, followed by intracranial injection of 10 µL of 100 µM CL-NC 1 hour later.
3.2.2 In vivo spinal cord imaging: Administer 10 µL of 100 µM Tz-IFDL via intrathecal injection, followed by intrathecal injection of 10 µL of 100 µM CL-NC 1 hour later.

4. Control Group Setup
4.1 Set up the following control groups:
4.1.1 Control Group 1: C57BL/6J mice receiving intracranial injection of tetrazine-free Compound 5 followed by CL-NC for brain imaging; C57BL/6J mice receiving intrathecal injection of tetrazine-free Compound 5 followed by CL-NC for spinal cord imaging.
4.1.2 Control Group 2: C57BL/6J mice receiving Tz-Br first, followed by CL-NC.
4.2 Positive controls are used to validate the experimental system; blank controls are used to eliminate fluorescence interference from reagents.

5. Detection and Analysis
5.1 Instruments:
5.1.1 Chemiluminescence imaging system, with the maximum emission wavelength of the CL-NC/Tz-IFDL reaction being 710 nm.
5.1.2 Fluorescence imaging system, with the emission wavelength range of probe fluorescence being 640-675 nm.
5.2 Result Analysis:
5.2.1 Changes in fluorescence intensity:
5.2.1.1 In vivo imaging: The chemiluminescence intensity of normal mice reaches a peak at 3 h, with brain imaging signals lasting > 8 h and spinal cord imaging signals lasting > 6 h; the peak intensity of AD model mice is lower than that of normal mice.
5.2.2 Fluorescence localization:
5.2.2.1 In vivo imaging: Chemiluminescence signals localize to the mouse cerebral cortex/hippocampus (brain imaging) and L4-L5 spinal canal (spinal cord imaging).
5.2.3 Color changes: The CL-NC/Tz-IFDL reaction produces near-infrared chemiluminescence (710 nm); the probe produces deep red fluorescence (640-675 nm).
5.2.4 Signal-to-noise ratio: In chemiluminescence mode, the maximum signal-to-noise ratio reaches 182 for brain imaging and 124 for spinal cord imaging, which is significantly higher than that in fluorescence mode.