Rat anti cd4 clone rm4 5

Tissue preparation was described previously in Reference [31 (link)]. Briefly, four micrometer-thick sections of Harderian and salivary glands were stained with HE. For immunohistochemical assessment, sections were deparaffinized, rehydrated, and then heated in 10 mM sodium citrate buffer (pH 6.0) for 20 min at 121 °C for antigen retrieval. Subsequently, sections were incubated with 0.3% H₂O₂ in methanol for inhibition of endogenous peroxidase for 30 min and incubated with primary antibodies diluted 1:30 in 1% bovine serum albumin in PBS (BSA-PBS) for 60 min at room temperature. Primary biotinylated antibodies included hamster anti-CD3ε (clone 145-2C11; BD Pharmingen), rat anti-CD4 (clone RM4-5; BD Pharmingen), rat anti-CD8α (clone 53-6.7, BD Pharmingen), which were detected by horse radish peroxidase (HRP)-conjugated streptavidin (Dako, Glostrop, Denmark), and rabbit anti-IgG1 (Novus Biologicals, Littleton, CO, USA), which was detected by EnVision FLEX (Dako). Immunoreactivity was visualized using 3,3′-diaminobenzidine substrate chromogen (Dako), counterstained with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). The slides were examined on Biorevo BZ9000 microscopy (Keyence, Osaka, Japan) and Vanox ADH (Olympus, Tokyo, Japan).

Fifteen microliters of blood were collected from the tail vein. Briefly, blood was lysed using 1X Mouse Lyse Buffer (R&D Systems) and washed twice with PBS. Cells were stained with rat anti-CD4 (clone RM4–5, BD Biosciences), rat anti-CD25 (PC61.5, eBioscience), rat anti-CD44 (IM7, eBioscience) antibodies. Intracellular staining of Foxp3 was done using Foxp3 staining buffer set (eBioscience) and rat anti-Foxp3 (FJK-16s, eBioscience) following manufacturer’s instructions. Data was acquired using BD LSRFortessa™ cell analyzer. Dead cells and duplets were excluded in all analyzes using forward and side scatter gating. Results were analyzed with FlowJo version X software (Tree Star, Inc).