Hpa029874

To co-localize cathelicidin with M. agalactiae, a double immunofluorescence stain was performed.
Three μm tissue sections were mounted on Superfrost™ slides (Thermo Scientific). Dewaxing, rehydration, and antigen retrieval were conducted in Dewax and HIER (heat-induced epitope retrieval) buffer L pH 6.2 (Thermo Scientific) at 98 °C for 20 min by means of an Electric Vegetable Steamer. Slides were then chilled in milli-Q water for 20 min and washed three times in PBST.
Tissues were blocked with Normal Horse Serum (NHS) (VECTOR) for 30 min and then incubated overnight at 4 °C with a rabbit hyperimmune serum raised against M. agalactiae rP48 (Rosati et al., 2000; (link)Cacciotto et al., 2016) (link). On the following day, the tissue sections were washed with PBS-T, incubated with a broad spectrum biotinylated antibody (Invitrogen) for 15 min, and the signal were revealed by incubating with streptavidin-Alexa Fluor 555 conjugate (Invitrogen), for 45 min at room temperature in the dark. After washing 3 times with PBS, the sections were subjected to a new nonspecific blocking step with NHS and incubated overnight at 4 °C with an anti-CAMP, rabbit polyclonal antibody (Sigma Aldrich HPA029874), diluted 1:1500. The signal was revealed as described above using streptavidin-Alexa Fluor 488 conjugate (Invitrogen).

To co-localize cathelicidin and cytokeratin, tissue sections were subjected to a double immunofluorescence, as described above, with slight modifications. The anti-CAMP, rabbit polyclonal antibody (Sigma Aldrich HPA029874) was used in the first incubation and it was revealed with streptavidin-Alexa Fluor 555 conjugate (Invitrogen). In the second round, tissues were incubated with α-cytokeratin (peptide 18) FITC conjugated (Sigma Aldrich F4772) diluted 1:100. Nuclei were counterstained with Hoechst, washed with Milli-Q water, and slides were mounted with Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich).