Dm4500b

Lung tissues were washed twice with PBS, fixed with 3.5% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Paraffin embedded 5-micron sections of all the tissue samples. Briefly, the tissue sections were deparaffinized in two changes of xylene for 5 min each, hydrated in two changes of 100% ethanol for 3 min each, changes of 95% and 80% ethanol for 1 min each, and finally washed in distilled water. The sections were processed for antigen retrieval in a water bath containing sodium citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) at 95–100 °C for 40 min, and then allowed to cool to room temperature for another 60 min. The sections were rinsed in PBS-Tween 20, twice for 2 min each. The sections were blocked with 2% BSA in PBS for 1 h at room temperature. The sections were then incubated with a 1:500 dilution of the homologous primary N-specific antiserum 3-NT (39B6; sc- 32757), IRβ (C-19; sc-711) and AKT (sc- 8312) in PBS for 1 h in a humidified chamber. After three washes in PBS, the sections were incubated with the secondary antibody rhodamine (sc-2095) or fluorescein (sc- 2989) for 30 min. After a further wash cycle, the sections were mounted with 4,6-diamidino-2-phenylindole (DAPI) and viewed under an immunofluorescence microscope (Leica DM 4500 B).

Formalin or formaldehyde fixed, paraffin-embedded tumor tissues were cut into 5 µm sections. The immunohistochemical staining was performed as described by Takacova and colleagues [40 (link)] using DakoCytomation EnVisio+ System-HRP (DAB). CAIX was stained by M75 antibody (hybridoma medium diluted 1:100) for 1 h at room temperature without antigen retrieval. Pictures were taken by a Leica DM4500B microscope with a Leica DFC480 camera.

Following fixation in 4% neutral buffered formalin, serial sections (5 μm) of paraffin-embedded specimens were stained with Masson’s trichrome. Digital images were generated of all specimens (Leica DM 4500B, Allendale, NJ, USA).

The quantitative determination of apoptosis in retinas was histologically assessed. For this purpose, animals were euthanized by intravenously administering sodium pentobarbital (100 mg/kg) and perfused with phosphate buffered saline (PBS) (Fisher Scientific, Madrid, Spain). Eyes were enucleated and kept in Davidson’s fixative (8% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA), 30% ethanol, 10% glacial acetic acid (Panreac Quimica S.L.U., Castellar del Vallés, Barcelona, Spain) for 24 h at 4 °C before being transferred to 70% ethanol until use. The paraffin-embedded retinas were sectioned at 5 μm by a microtome (Leica Biosystems, Wetzlar, Germany). Apoptosis was detected using the In Situ Cell Death Detection Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, and was then examined by microscopy (Leica DM 4500B, Wetzlar, Germany). Images were analysed by the free NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Dissected tumours were formalin-fixed and embedded in paraffin according to the standard histological procedure. Because of melanin presence in tissues, sections were bleached using potassium permanganate solution (2 g/l) for 20 min at 65 °C. After rinsing in PBS, sections were immersed in 1% oxalic acid for 1 min at room temperature followed by rinsing in PBS for 10 min. Immunohistochemistry was performed on Dako Autostainer employing DakoCytomation EnVision®+ System-HRP (DAB) for use with Mouse Primary Antibodies according to the manufacturer’s instructions. The sections were labelled with M75 antibody in hybridoma medium diluted 1:100 for 1 h at room temperature and after washing incubated with secondary anti-mouse-HRP antibody for 30 min at room temperature. Absence of nonspecific binding of the secondary antibody was confirmed on parallel sections by omitting the primary antibody. Staining was visualised 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 Millipore, MA, USA). The stained sections were examined using a Leica DM4500B microscope and images were captured by a Leica DFC480 camera.

As we previously described,⁴⁰ the temporal bones were fixed in 4% PFA, and the basilar membrane was then carefully dissected and cut into three separate fragments: apical, middle and basal turn. The fragments were permeabilized in 2% Triton X-100, stained for F-actin with phalloidin (Invitrogen), and observed with a fluorescence microscope (Leica DM4500 B). Hair cells were counted as being present if cell bodies and the V-shaped hair bundles were intact.