Roti mount

To visualize the activation of reporter lines, the trachea of 3^rd instar larvae were dissected in cold PBS and mounted in Roti-Mount (Roth, Karlsruhe, Germany) to stain the DNA. Images were acquired using a Zeiss Axio Imager Z1, equipped with an Apotome and an AxioCam MRm camera system (Zeiss, Oberkochen, Germany).

5 µm paraffin sections were deparaffinized, incubated with hematoxylin (Sigma-Aldrich) for 1 min, followed by a short rinse in tap water and 1% HCl. Bluing was performed under running tap water for 10 min. Afterwards, 1% eosin solution (Carl Roth) was incubated for 1 min, sections were dehydrated and mounted with Roti®-Mount (Carl Roth).

For the immunohistochemistry staining the paraffin placenta sections were dewaxed at first for 20 min in Roticlear and washed in 100% ethanol. To block endogenous peroxidases, sections were incubated for 20 min in 6% H₂O₂ (Roth, Karlsruhe, Germany). Then they were dehydrated in 100%, 70%, and 50% ethanol to destilled water. For protein unmasking, the slides were cooked with sodium-citrate-buffer. The slides were blocked at first with ZytoChem-Plus HRP Polymer Kit (Mouse/Rabbit) Reagent 1 (Zytomed Systems GmbH, Berlin, Germany). The primary antibody CD68 (Sigma Aldrich, St. Louis, MO, USA) was diluted in PBS, put on the sections and incubated in the fridge at 4 °C for 16 h. Then they were treated with ZytoChem-Plus HRP Polymer Kit Reagent 2 and with ZytoChem-Plus HRP Polymer Kit Reagent 3. For the enzymatic reaction with the peroxidase contained in the HRP Polymer, the slides were incubated with DAB (Aligent Technologies, Santa Clara, CA, USA) for 2 min and the stopped with aqua dest. The nuclei were stained with Mayer’s acidic hemalaun (Waldeck, Münster, Germany) for 2 min and blued in tap water. For final dehydration, an ascending alcohol series was followed with 70%, 90% and 100% ethanol to Roticlear and subsequent coverslipping of the slide with Roti-Mount (Roth, Karlsruhe, Germany) coverslip medium. A negative control was also performed with an IgG antibody.

To examine if M. gallisepticum infection of TOC affects mucus production, visualization of acidic epithelial mucin via Alcian blue staining was performed. Samples were prepared as described above. The following procedure is based on the study from Crespo-Moral et al. [39 (link)] and was modified according to H&E staining procedures. Paraffin was removed, and samples were hydrated in xylene, descending alcohols, and distilled water. Afterwards, sections were stained with Alcian blue (Imperial Chemical Industries Ltd) for 20 min. After the last two washing steps, sections were dehydrated with an ascending alcohol series and mounted with Roti^®Mount (Carl Roth GmbH + Co.KG). Goblet cells were counted for three microscopic fields (400-fold magnification), and the average number/ring was calculated. The group average was determined with five sections/group/time point.

Rehydrated tissue sections were soaked in Mayer’s hematoxylin (Gatt-Koller, Absam, Austria) for 3 min, followed by a 10 min rinse under warm, running tap water. A quick wash in distilled water and 95% ethanol was followed by eosin staining (Gatt-Koller) for 1 min. Stained sections were dehydrated by an increasing series of alcohol concentrations, which were finished by a double wash in Roticlear. Finally, sections were mounted with a non-aqueous mounting media (Rotimount, CarlRoth).