Shandon varistain gemini slide stainer

Larvae were anesthetized as described above and fixed overnight in aqueous Bouin’s fixative (RICCA Chemical, Pokomoke City, USA). Larvae were washed in 70 % EtOH until supernatant was clear (at least six washes of 1 h each at room temperature). Larvae were oriented in Richard Allan Scientific HistoGel (ThermoFisher, Waltham, USA). The HistoGel discs containing oriented larvae were fixed for 1 h in Bouin’s fixative, and subsequently washed in 70 % EtOH until supernatant was clear. Samples were infiltrated with paraffin using a Tissue-Tek VIP5 Vacuum Infiltration Processor (Sakura Finetek, Torrance, USA; model 5215). Sections were cut at 4–5 μm and slides were stained with Harris acidified hematoxylin (3 min) and eosin Y (10 s) using the “standard” H&E protocol [35 ] on a Shandon Varistain Gemini Slide Stainer (ThermoFisher, Waltham, USA). Slides were imaged using an Axioplan 2 (Zeiss, Oberkochen, Germany) equipped with a Q-Capture MicroPublisher 5.0 RTV camera/software (QImaging Scientific, Surrey, Canada) using 10× and 40× air objectives and 63× and 100× oil-immersion objectives.

A complete post mortem examination was performed on all mice and all abnormalities registered. Spleen, liver, lung and femurs/tibias were immediately fixed in 4% formaldehyde solution, buffered at pH6.9 (Merck Chemicals Ltd, Belgium) and processed in automated Shandon Citadel 1000 (Thermo, Belgium) for paraffin embedding, with previous decalcification for femurs and tibias. All samples were sectioned in 4μm sections with Shandon Finesse Microtome (Thermo, Belgium) and Hematoxylin and Eosin (H&E, both Sigma-Aldrich, Germany) staining was performed on tissue sections with Shandon Varistain Gemini Slide Stainer (Thermo, Belgium). Pictures were analyzed and captured with CellaVision DM96 automated microscope. Blind analysis was carried out by a clinical pathologist. In addition, the skeletons were analyzed by radiography to assess lytic lesions. X-rays were performed in the Centre Hospitalier Luxembourg (CHL) radiology department, on the breast x-ray scanner Microdose Spectra (Philips, Germany).
From all femurs, tibias, humerii and radii (except the ones fixed in formalin), bone marrow was flushed out. Cells were collected in RPMI1640 medium and filtered through 100μm filters (ImmunoTools, Germany), washed and centrifuged at 200g to get single cell suspensions. Bone marrow cells were used for protein, flow cytometry analysis and drug-resistance testing.

After being deparaffinized in xylene and absolute ethanol with the Shandon Varistain Gemini Slide Stainer (Global Medical Instrumentation Inc., Ramsey, MN), tissue sections were placed in a box filled with the appropriate epitope damasking buffer (10 mM citrate buffer/1 mM EDTA pH 7,5 for anti-IL-17A, anti IL-17E, anti-tryptase and anti-CD3 antibodies; 10 mM Tris buffer/1 mM EDTA pH 9 for anti-IL-17C and anti-IL-17F antibodies), and heated in a microwave at 600 W for 20 min. Endogenous peroxidase activity was inhibited by treatment with 0.9% hydrogen peroxide in water for 20 min at room temperature. The tissue sections were incubated for 1 h with primary antibodies diluted in PBS-0.3% BSA, washed twice with PBS and incubated for 30 min with biotinylated secondary antibodies diluted in PBS-0.3% BSA. Immune reactivity was detected with the Vectastain ABC kit using DAB as substrate. Subsequently, slides were counterstained with hematoxylin using the Shandon Varistain Gemini Slide Stainer and mounted with the ClearVue Coverslipper (Thermo Scientific, Waltham, MA). Immunohistochemical stainings were acquired with the Mirax-midi scan microscope (Carl Zeiss Microscopy GmbH, Oberkochen, DE). In negative controls primary antibodies were omitted.