Digital sight ds u3 camera

Nuclear area was calculated on the basis of the images of DAPI staining, taken with Nikon Eclipse fluorescent microscope and Nikon Digital Sight DS-U3 camera (Nikon, Tokyo, Japan). Analysis was performed with the use of ImageJ software.

Detection of SA-β-Gal was performed according to Dimri et al. [29 (link)]. Briefly, cells were fixed with 2% formaldehyde, 0.2% glutaraldehyde in PBS, washed and exposed overnight at 37 °C to a solution containing: 1 mg/mL 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2 and 0.02 M phosphate buffer, pH 6.0. All of the chemicals were purchased from Sigma Aldrich. Photos were taken in transmitted light using the Nikon Eclipse Ti-U fluorescent microscope and Nikon Digital Sight DS-U3 camera (Nikon, Tokyo, Japan).

After dissection, the muscles were weighed (Supplementary Table S1) and immediately mounted in an optimal cutting temperature compound (6769006, Thermo Fisher Scientific) and frozen in isopentane, previously cooled in liquid nitrogen. Cryo-sectioning was performed on a Leica cryostat to generate 10 µm sections. After that, muscle cross-sections were air-dried for 30 min and stained with Mayer’s Hematoxylin Solution (MAS16-500ML, Sigma-Aldrich) for 30 min. After rinsing with tap water for 15 min, the sections were stained with Eosin Y Solution Counterstain (HT110216-500ML, Sigma-Aldrich) for 7 min followed by dehydration through 2 changes each of 95% reagent alcohol and xylene for 2 min. After that, the sections were mounted with a resinous mounting medium (HX74511279, Merck). Morphometric analysis was performed on digital muscle cross-sections taken by Nikon Eclipse Ti-U microscope equipped with Nikon Digital Sight DS-U3 camera (Nikon Corporation, Shinagawa, Tokyo, Japan). Analysis of muscle cross-section area, percentage of fibers with appropriate diameter, number of fibers per selected area as well as a number of nuclei per myofiber was carried out on 3–5 sections from three animals in each group, using ImageJ software (Schindelin et al., 2012 (link)).

SA-β-Gal activity detection assay was carried out according to Dimri et al. [37 (link)]. At appropriate time points, the cells were fixed in 0.2% glutaraldehyde in PBS, 2% formaldehyde, and then washed and incubated overnight at 37 °C with a mixture of 5 mM potassium ferrocyanide, 150 mM NaCl, 2 mM MgCl₂, 0.02 M phosphate buffer, pH 6.0 and 1 mg/mL 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (all agents from Sigma-Aldrich). Results were visualized using a Nikon Eclipse Ti-U microscope and Nikon Digital Sight DS-U3 camera (Nikon, Tokyo, Japan) images were taken in a transmitted light.

Whole mount in situ hybridizations were performed as previously described^{38 (link)}. Briefly, larvae of different developmental stages (24 hpf, 72 hpf and 120 hpf) were fixed overnight in 4%PFA in PBS. At minimum 20 embryos were used for each timepoint. Antisense RNA probes were generated by means of polymerase chain reactions, including T7 promoters in the primers from a mix of 24 hpf, 48 hpf and 72 hpf cDNA and subsequent transcription with T7 Polymerase (Roche). Primer sequences used are found in Supplementary Table S1. Images of the embryos were taken with a Nikon Digital Sight DS-U3 camera and Elements software (Nikon). Image adjustments for brightness and contrast were done with Adobe Photoshop and figures were assembled using Adobe Illustrator. 120 hpf embryos, grown without PTU, were euthanized using MS222 and subsequently fixed in 4% paraformaldehyde for 24 h. Fixed embryos were treated in 10% and subsequently 30% sucrose for 24 h each. Post sucrose washes, the embryos were mounted in OCT (optimal cutting temperature) embedding medium (Sakura) and frozen in cryosection molds. Cryosections were collected at 10 µm thickness using a Leica cryotome. Images were collected using a Nikon Ti2 compound microscope with a 20X NA 0.95 objective. Subsequent processing was performed using Adobe Photoshop and Illustrator.

Separately, petrological thin sections of rocks and eggshells were prepared using standard procedure [52 ]. It is important to mention that rock and eggshell specimens were collected from numerous clutches barring a few clutches owing to lack of well preserved material and hostility from neighbouring villagers. The collected rock specimens were initially cleaved and soaked in resin for consolidation. However, this exercise was avoided in the case of eggshell specimens to avoid damage caused to specimens by the hot resin. The specimens were ground and polished using carborundum powder of various grades (400–1000 μm). After making the radial section of the eggshells flat, the flattened surfaces were pasted on glass slides using araldite and were left to dry for 12–24 hours. They were ground and polished to achieve a thickness of 0.03 mm and the final step in grinding was completed with diamond polishing. A total of forty-six eggshell thin sections were examined under the petrological microscope to study the eggshell morphology. Photomicrographs of the thin sections were taken using Carl Zeiss Axio Imager A1m High Resolution Petrological Microscope in the Vertebrate Palaeontology Laboratory and Nikon Eclipse 50i Polarizing Microscope with Nikon’s Digital Sight DS-U3 camera attachment in Metamorphic Petrology Laboratory of the Department of Geology, University of Delhi.