Polyclonal rabbit anti mouse biotinylated secondary antibody

Cytospins of SVF cells isolated from MAT and SAT as well as leukocytes isolated from whole blood of dairy cattle were prepared using 1 to 3 × 10⁵ cells. The slides were then methanol fixed and stained for TCR γδ by a previously described protocol with some modifications^{20 (link)}. Briefly, peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol (Merck, Darmstadt, Germany) for 20 min. Slides were then incubated in a moist chamber for 20 min with normal rabbit serum (Dako, Glostrup, Denmark) diluted 1:5 in 10% BSA (Sigma), to eliminate non-specific staining. Excess serum was removed and the slides were incubated in a moist chamber overnight at 4 °C, with a monoclonal mouse anti-bovine TCRγδ (clone GB21A) diluted 1:100. Slides incubated with anti-TCRγδ antibody were washed and incubated for 30 min at room temperature with the polyclonal rabbit anti-mouse biotinylated secondary antibody (Dako) diluted 1:200 and then with the avidin–biotin peroxidase complex (Dako), for a further 30 min. The colour in all slides was developed by incubation with 3,3′-diaminobenzidine (Dako). After counterstaining sections with Mayer’s Haematoxylin (Merck), slides were mounted in Entellan (Merck). A positive reaction was indicated by the presence of brown cytoplasmic staining.

The protein expressions of TLR2, TLR4, MyD88, and NF-κBp65 were detected according to a method described previously [3 (link)]. Briefly, 4 μm colon sections were first treated with 3% hydrogen peroxidase for 10 min to block endogenous peroxidase, then incubated with the polyclonal primary antibody of TLR2, TLR4, MyD88, and NF-κBp65 (diluted to 1 : 100) overnight at 4°C. The colon sections were then washed with phosphate-buffered saline (PBS) and incubated with polyclonal rabbit anti-mouse biotinylated secondary antibody (Dako, CA, USA). After that, colon sections were incubated with 3,3′-diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA) and then stained with hematoxylin. Finally, images were observed under an Olympus BH-2 microscope (Tokyo, Japan).