May gr nwald and giemsa stain

In the acute protocol, all animals were sensitized to ovalbumin as described above, and received a single saline or ovalbumin challenge 5 weeks after sensitisation (Fig. 1a). Animals were treated with different dosages of tiotropium (0.01, 0.03, 0.1 and 0.3 mM, 3 min inhalation time), ciclesonide (0.001, 0.01, 0.1 and 1 mg/kg) or the combination of tiotropium and ciclesonide (0.1 mM and 1 mg/kg, respectively) before ovalbumin challenge (Additional file 1: Table S1). Twenty five hours after the ovalbumin challenge, animals were anaesthetized with 20 mg/ml Brietal-sodium, 35 mg/kg ketamine hydrochloride and 6 mg/kg Sedamun intraperitoneally, which ensured a fast, deep anaesthesia. The lungs were gently lavaged with 5 ml of sterile saline at 37 °C using a tracheal cannula, followed by three subsequent aliquots of 8 ml of saline. The recovered samples were placed on ice and centrifuged at 290 g for 10 min at 4 °C. The combined pellets were resuspended to a final volume of 1.0 ml in PBS, and total cell numbers were counted using a coulter counter (Casy Rock). For cytological examination, cytospin preparations were stained with May-Grünwald and Giemsa stain (Sigma Chemical, St. Louis). A cell differentiation was performed by counting at least 400 cells in duplicate.

The total number of inflammatory cells present in the BALF was determined via hemocytometer (FastRead102, Immune Systems LTD, United Kingdom) following staining with Turk’s solution (Histolab Products AB, Gothenburg, Sweden). 50,000 cells per sample were plated on glass slides by cytospin centrifugation. To visualize inflammatory cells, the slides were stained with May-Grünwald and Giemsa stain (Sigma-Aldrich). Differential cell counts were performed by counting 300 cells from BALF per mouse.