Vectastain abc kit elite kit

Tissues for microscopic analysis were fixed overnight in 20% buffered formalin and transferred to 70% ethanol the next day. A Tissue-Tek VIP autoprocessor (Sakura, Torrance, CA) was used to process samples for paraffin-embedding. Tissue blocks were then sectioned to 4 μm, sections mounted on glass slides. Hematoxylin and Eosin (H&E) staining was performed at the University of California, Davis, Dept. of Pathology and Laboratory Medicine. Immune cells were stained with specific antibodies based on the manufacturer’s instructions. Anti-mouse CD8 (1:500 dilution; 14–0808, eBiosciences) was used as CD8 marker. Anti-mouse CD68 (1:700 dilution; PA1518, Boster) was used to stain macrophages. Anti-mouse OX40 antibody (1:2000 dilution; ab229021, abcam) was used to label the activated T cells. Anti-mouse CD206 (1:2000 dilution; ab64693, abcam) was used as a marker for M2 macrophages. Detection of the primary antibodies was performed using the appropriate isotype specific species secondary antibody with the Vectastain ABC Kit Elite Kit and a diaminobenzidine Peroxidase Substrate Kit (Vector Labs, Burlingame, CA) for amplification and visualization of signal. Stained slides were scanned on an AT2 Scanscope (Leica Biosystems), and digital images viewed using the Imagescope software.

Paraffin blocks were sectioned and mounted on glass slides. All tissue sections were subjected to antigen retrieval which was performed for 45 minutes using a citrate buffer at pH 6.0 in a Decloaking Chamber (Biocare Medical, Concord, CA) at 125°C and 15 psi. Tissues were blocked with normal goat serum, then incubated with polyclonal rabbit antimurine SPL (1:1000)^{16 (link)} primary antibodies overnight at room temperature in a humidified chamber, followed by a biotinylated goat antirabbit secondary antibody (1:1000). The Vectastain ABC Kit Elite Kit and a diaminobenzidine Peroxidase Substrate Kit (Vector Labs, Burlingame, CA) were used for amplification and visualization of signal, respectively. Slides were scanned as described above.