Dm4500b microscope

The spheroids were fixed in Carnoy fixative (Ethanol:Chloroform:Glacial acetic acid 6:3:1) and embedded in paraffin. Patient tumour tissues were fixed in neutralized formalin and embedded in paraffin. Antigen retrieval was performed at low pH retrieval buffer (EnVision^® Flex Target Retrieval Solution Low pH) at PT-Link (Dako). Immunohistochemical analysis was performed on an automated immunostainer (Dako Autostainer) using DakoCytomation EnVision^®+ System-HRP (DAB) according to the manufacturer’s instructions. For CA IX staining, the sections were labelled with M75 antibody (hybridoma medium) diluted 1:100 for 1 h at room temperature. For β1 and β2 adrenoreceptors and Ki-67 the sections were incubated with anti β1 (Invitrogen, Carlsbad, CA, USA, 1:500), anti β2 (Abcam, 1:250) overnight at 4 °C, for Ki-67 (DAKO, 1:100) for 1h at room temperature and, after washing, incubated with secondary anti mouse-HRP or anti rabbit-HRP antibody for 30 min at room temperature. Staining was visualized using 3,3’-diaminobenzidine (DAB) as a chromogenic substrate for 1 min. The sections were counterstained with Mayer’s haematoxylin and mounted in Aquamount (Merck, Darmstadt, Germany). The stained sections were examined using a Leica DM4500B microscope and images were captured by a Leica DFC480 camera.

The distribution of heterochromatin was analyzed by Giemsa C-banding according to Sumner (1972) (link), after treatment with 0.2M HCl for 10 min at room temperature, Ba (OH)₂ for 1 min and 40 s at 60 °C, and 2× SSC for 1 hour at 60 °C. The AT-rich bands were detected with

4’-6-diamino-2-phenylindole

(DAPI), respectively, according to Schweizer et al. (1983) . The slides were stained with 2µg/mL DAPI for 30 min. Slides were mounted with a medium composed of glycerol/McIlvaine buffer (pH 7.0) 1:1, plus 2.5mM MgCl₂. All images were acquired with a Leica DM 4500 B microscope, equipped with a DFC 300FX camera and Leica IM50 4.0 software, and optimized for best contrast and brightness with iGrafx Image software.

Fluorescence in situ hybridization

(FISH) followed the methods described by Pinkel et al. (1986) with an 18S rDNA probe obtained from Prochilodusargenteus Spix & Agassiz, 1829 (Hatanaka and Galetti Jr. 2004 (link)). The 18S rDNA probe was labeled with biotin-14-dATP (Roche Applied Science) by nick translation and the 5S rDNA probe from Leporinuselongatus Linnaeus, 1758 (Martins and Galetti Jr. 2001 (link)) was labeled with digoxigenin 11-dUTP (Roche Applied Science) by PCR. The hybridization signal was detected using avidin-FITC (fluorescein isothiocyanate) (Life Technologies) for the 18S rDNA probe and anti-digoxigenin-rhodamine (Roche Applied Science) for the 5S rDNA probe. The chromosomes were counterstained with propidium iodide or DAPI, respectively. All the images were acquired with a Leica DM 4500 B microscope equipped with a DFC 300FX camera and Leica IM50 4.0 software and optimized for best constrast and brightness with Adobe Photoshop CS6 software.

Paraffin-embedded tissues were sectioned and mounted onto glass slides. The 5 μm-thick slides were deparaffinized and rehydrated. CAIX was detected using the DAKO EnVision™ FLEX System according to the manufacturer’s instructions. Sections were incubated with anti-CAIX antibody M75 (1:25) over night at 4 °C. Negative controls were prepared by omission of the primary antibody. The stained sections were examined using a Leica DM4500B microscope and images were captured with a Leica DFC480 camera.

0.3% hydrogen peroxide for 10 min at room temperature was used to remove endogenous peroxidase activity and blocking serum for 30 min at room temperature. After primary antibody incubation with anti-mouse/rabbit serum (DAKO EnVision - TM Peroxidase, Rabbit, Dako Corporation, Carpinteria, CA) for 30 min at room temperature, 3,3=-diaminobenzidine (Sigma- Aldrich, Milan, Italy) was used. Counterstained with hematoxylin was performed. Negative controls were also done. Pictures were taken with a Leica DM4500B microscope coupled with a DFC320R2 camera for image acquisition and analyzed using Fiji software Color deconvolution/H DAB to analyze the signal of CD31. Threshold software was used to quantify area with positive CD31 signal, data were expressed as percentage of tissue covered per field.

Spheroids were fixed in 4% PFA and embedded in paraffin. Antigen retrieval was performed at low pH EnVision^® Flex Target Retrieval Solution. A Dako EnVision FLEX+ detection system was used for immunohistochemistry staining. Antibodies are detailed above. Stained sections were examined using Leica DM4500B microscope. For CAIX immunofluorescence staining sections were labeled with M75 antibody conjugated with anti-mouse Alexa 555 antibody (Abcam, Cambridge, UK, Unab274040). The nuclei were stained with DAPI (300 nM). After washing the spheroids were analyzed using confocal microscopy LSM510 Meta (Carl Zeiss, Jena, Germany), 20× objective.