*4.4. Immunohistochemistry (IHC)*

Sagittal six-micrometer sections from injured and uninjured samples from both age groups of C57Bl/6J mice were used for IHC. Unitrieve was used as an antigen retrieval method for 30 min at 65 ◦C. Primary antibodies: CYTL1 (Proteintech, Rosemont, IL, USA; 15856-1-AP(1:75)), PLOD2 (Invitrogen, Carlsbad, CA, USA; PA5-69194(1:100)), and CHAD (Invitrogen, Carlsbad, CA, USA; PA5-53761(1:300)) were used and incubated overnight at room temperature in a dark, humid chamber. Negative control slides were incubated with secondary antibody-only. Stained slides were mounted with Prolong Gold with DAPI (Molecular Probes, Eugene, OR, USA). Slides were imaged using a Leica DM5000 microscope. ImagePro Plus V7.0 Software and a QIClick CCD camera (QImaging, Surrey, BC, Canada) were used for imaging and photo editing.
