Quantifying T-cell Subtypes in Spinal Cord Tissue

The cross-sections (5 mm thick) were dewaxed using xylene and dehydrated in a graded series of alcohols after incubation at 60°C for 1 h. The endogenous peroxidase activity was quenched with 3% H₂O₂, and heat-induced epitope retrieval was done in sodium citrate buffer. Sections were incubated with anti-CD3 antibody (Abcam, Cambridge, UK), anti-CD4 antibody (Abcam, Cambridge, UK), and anti-CD8 antibody (Abcam Cambridge, UK) overnight at 4°C, followed by incubation with SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (Cell Signaling Technology, Danvers, MA, USA) according to instructions from manufacturers. Final color product was developed with SignalStain DAB Substrate Kit (Cell Signaling Technology, Danvers, MA, USA), and then sections were counterstained with hematoxylin (Leagene, Beijing, China). Images were captured by LEICA DM6000B with a LEICA DFC300 FX (Leica Microsystems Ltd., Solms, Germany) at a magnification of 200x. Six fields were evaluated for each slide [25 (link)]. The numbers of positive cells per mm² of spinal cord tissues were made by manual counting at Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) [26 (link)].