Flexacam c1

The adult specimens examined in this study were obtained in October, 2021 from larvae collected in September, 2021 on inflorescences of Glandulariagynobasis (Wedd.) N. O’Leary & P. Peralta (Verbenaceae) in Cuesta El Águila (18°29'08"S, 69°51'55"W), at about 1950 m elevation in the Cardones ravine, Arica Province, northern Chile. Two larvae were kept in 95% ethanol at –20 °C until DNA extraction. The abdomen of each adult was removed and placed in hot KOH 10% for a few minutes for genitalia dissection. Genitalia were stained with Eosin Y and Chlorazol Black and mounted on slides with Euparal. The photo of the adult was taken with a Leica Flexacam C1 digital camera attached to a Leica M125 stereomicroscope. Photos of the genitalia were taken with a Leica MC170 HD digital camera attached to a Leica DM1000 LED light microscope. Each image was constructed with about 5–20 photos assembled with the software Helicon Focus 8. Terminology of Huemer and Karsholt (2010) (link) is followed for the genitalia. The specimens studied are deposited in the “Colección Entomológica de la Universidad de Tarapacá” (IDEA), Arica, Chile.

After dietary supplementation with normal (30 ppm) and increased (150 ppm) zinc for 6–8 weeks, WT and Tbr1^+/− mice were euthanised with CO₂ and transcardially perfused with 20 mL of ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). The brains were collected and postfixed in 4% PFA for 24 h at 4 °C and then incubated in 30% sucrose in 0.1 M phosphate buffer for 72 h at 4 °C. Coronal brain sections were prepared at 50 µm thickness. Brain sections from 7 animals were used for each group (i.e. WT_30ppm vs. Tbr^+/−_30 ppm vs. WT_150ppm vs. Tbr^+/−_150 ppm). Brain sections were placed in alcohol/chloroform solution and then rinsed with 70% ethanol, 5 min × 3 times. The sections were then placed in 0.1% Luxol fast blue solution overnight at 56 °C and were destained with 0.05% lithium carbonate solution. Each whole brain section was imaged with a Leica MZ6 stereomicroscope using a Leica Flexacam C1 camera at 1.6X magnification.

For the examination of the immunohistochemically stained cervical cancer slides, a Leitz Diaplan photomicroscope (Leitz, Wetzlar, Germany) was used. The staining of each specimen was quantified by the application of the semi-quantitative immunoreactive score (IRS), which is used for the optical evaluation of the intensity and distribution pattern of antigen expression [42 (link)]. The IRS was calculated by multiplying the number of positively stained cells (in %) (0: no staining; 1: 1–10% stained tumor cells; 2: 11–50% stained tumor cells; 3: 51–80% stained tumor cells; 4: >80% stained tumor cells) with the predominant staining intensity (0: none; 1: weak; 2: moderate; 3: strong). The scale goes from 0 (no expression) to 12 (very high expression). Images were taken with Flexacam C1 (Leica Microsystems (Switzerland) Ltd., Heerbrugg, Switzerland).

The immunohistochemically stained cervical cancer specimens were examined using a Leitz Diaplan photomicroscope (Leitz, Wetzlar, Germany). Quantification of RIPK1, RIPK3, and pMLKL expression was performed by applying the semiquantitative immunoreactive score (IRS) which evaluates the intensity and distribution pattern of antigen expression (37 ). The IRS is calculated by multiplying the number of positively stained cells (in %) (0: no staining; 1: 1% - 10% stained tumor cells; 2: 11% - 50% stained tumor cells; 3: 51% - 80% stained tumor cells; 4: > 80% stained tumor cells) with the predominant staining intensity (0: none; 1: weak; 2: moderate; 3: strong). The scale goes from 0 (no expression) to 12 (very high expression). Images were taken with Flexacam C1 (Leica Microsystems (Switzerland) Ltd., Heerbrugg, Switzerland).

Images were acquired by Leica DM 2000 microscope (Leica Microsystems, Wetzlar, Germany), with 60 × magnification objective lens in combination with a Leica Flexacam C1 (Leica Microsystems, Wetzlar, Germany) 12-MP stand-alone microscope camera and captured by Software On-screen display (OSD) for stand-alone operation LAS X for Windows. Image manipulation (adjustments of brightness, contrast and color balance) were applied when needed to the entire image.