Rabbit anti cd4

For histologic examination, intradermal challenges on the abdomen skin were inducted on Day 40. After 20 min, 24 h and 48 h, the mice were sacrificed using cervical dislocation, and the challenge site was removed and placed in 10% neutral buffered formalin overnight at room temperature. The skin tissue was dehydrated gradually through 70%, 80% and 95% and absolute ethanol (Guo et al., 2020 (link)), then soaked in xylene for 1 h at 55–60°C followed by embedding in paraffin for 1 h. The specimens were sectioned to a thickness of 5 µm using a Leica RM2235 microtome (Leica) and followed by deparaffinisation and haematoxylin and eosin stain. The sections were also stained with rabbit anti‐CD4 (1:200) and rat anti‐CD8 (1:200) monoclonal antibodies (NovusBiologicals) to detect cluster of differentiation (CD)‐maker positive cells: The sections were incubated with peroxidase‐labelled goat anti‐rabbit and goat anti‐rat antibodies and stained in the substrate solution containing 3,3′‐diaminobenzidine (ZSGB‐BIO). Inflammatory cell infiltration was examined using light microscopy, and the images were taken using the microscopic camera system BA400 Digital (Motic Group Co. Ltd.).

Immunofluorescent staining was used to observe the infiltration of Th17 cells into the cerebral cortex. The rats were anesthetized and perfused using PBS (0.1 M) after 72 h following CA/CPR. The intact brains were removed and fixed with 4% paraformaldehyde for 12 h and then were dehydrated for 24 h in sucrose (20%) followed by sucrose(30%) for another 24 h at 4 °C. The sections of the brain were made by a freezing microtome and permeabilized for 30 min using 0.3% Triton X-100 and blocked with 1% BSA solution for 2 h. The brain sections were incubated with primary antibodies to goat anti-NeuN (1:500, Novus, USA), rabbit anti-CD4 (1:100, Novus, USA), and mouse anti-IL-17a (1:100, Santa Cruz, USA) overnight at 4 °C. The sections were washed with PBS 3 times and incubated with a secondary antibody. Secondary antibodies conjugated with Alexa Fluor 647 (donkey anti-goat, 1:1000, Abcam, USA), Alexa Fluor 594 (donkey anti-mouse, 1:1000, Abcam, USA), and Alexa Fluor 488 (donkey anti-rabbit, 1:1000, Jackson ImmunoResearch, USA) for 1 h at room temperature and then stained with DAPI (1:1000, Roche, Switzerland) at 24 °C for 8 min. Stained brain sections were washed by PBS 3 times and imaged with a confocal laser scanning microscopy (Nikon, Japan)