Lumar v12 stereomicroscope

The mice were given an overdose of sodium pentobarbital, and the back skin was removed and subsequently fixed overnight in 4% PFA at 4°C and then placed in increasing sucrose solutions at 4°C each overnight (10%, 20%, and 30% sucrose) before being snap frozen in blocks of OCT in liquid nitrogen‐cooled isobutane and stored at −80°C. The skin was sectioned at 30‐μm thicknesses using a Leica CM1950 cryotome (Leica Biosystems, Buffalo Grove, IL, http://www.leicabiosystems) for immunohistochemical analysis (n = 24 sections per animal; 3 animals for each condition). The grafts were fixed overnight in 4% PFA at 4°C then changed to phosphate‐buffered saline (PBS) overnight at 4°C. The total number of GFP⁺ HFs was counted using a Zeiss Lumar V12 Stereo microscope, and higher magnification imaging was done with a Zeiss Observer microscope using Axiovision software (Carl Zeiss Microscopy).

In the case of bright field microscopy, a Zeiss M-2 microscope was applied and images were taken using Zen software. For fluorescence microscopy, a Zeiss Lumar.V12 stereomicroscope was applied to record the images of cerebellar sections using AxioVision 4 software. For high magnification, fluorescence microscopy a Zeiss Z2 Imager with Zen software was used to record the images. After cropping the images, they were corrected for brightness and contrast, and finally assembled into montages using Adobe Photoshop CS5 Version 12.

After 5 days, the ECT were imaged from the side and top with a Lumar.V12 stereo microscope (Zeiss). The diameters of the ECT were measured at several positions and the cross-sectional areas (CSA) were calculated assuming an elliptical shape of the tissues (Santos et al. 2021 ).

All embryos were dissected in cold PBS and fixed for 20 minutes in 4% paraformaldehyde at 25°C. Following imaging of embryos with a Zeiss Lumar V12 stereomicroscope, samples used for tissue analysis were processed for either cryosection or paraffin embedding. Cryo-embedded samples were flash frozen in O.C.T. compound and sectioned to obtain 8 to 10μm thick sections as described (28 (link)). Paraffin-embedded samples were processed and embedded by the GCI Histology Core Facility and 6μm serial sections were obtained.

Homozygous UAS-C4 flies were crossed to ey-GAL4/CyO flies. F1 progeny were then selected based on their wing phenotype; straight winged flies expressed RNAi to deplete Nopp140, while curly-winged siblings served as a control. Flies were imaged using a Lumar.V12 SteREO microscope (Zeiss, Jena, Germany) fitted with an Axiocam MRc5 camera (Zeiss, Jena, Germany). To measure eye size, a line was drawn around visible ommatidia using the contour (spline) function on the ZEN 2 Pro Software (Zeiss). The data are presented as average eye size (measured in μm²) per genotype.

To determine whether any difference existed in the external morphological characteristics of the maxillofacial structures and limbs, both the malformed and normal fetuses and NBs of DRs and CRs were analyzed using a Lumar V12 stereomicroscope (Zeiss), with special emphasis on the development of the eyelids, ears, and interdigital webbing. Images were captured using an Axiocam MRc microscope digital camera (Zeiss). In addition, 10 forelimbs and 10 hind legs from each group, previously fixed in 4% formalin, were processed to determine their histological characteristics. These specimens were made transparent with cedar oil and embedded in VIP (Sakura Finetek, Torrance, CA, USA) paraffin. Serial 5-μm thick histological sections in the sagittal plane were prepared along the palmar and plantar surfaces and stained with Hematoxylin and Eosin. An equal number of limbs were fixed in 2.5% glutaraldehyde and processed for analysis with a scanning electron microscope. After dehydration in a graded series of alcohols, these samples were prepared via critical point drying in a Samdri 789^a (Tousimis Rockville MD) apparatus and shadowed with a 350-nm thick gold layer in a Denton Vacuum Desk 1A apparatus (Cherry Hill Industrial Centre, NJ, USA). The samples were observed at 15 kV in a JEOL JSM 5300 scanning electron microscope (JEOL, Tokyo, Japan).

To label proliferating cells, pregnant dams mated with Fgf20^-/- males were given one intraperitoneal injection of 25 mg/kg body weight EdU (Life Technologies, Eugene, OR) dissolved in saline 2 hr prior to sacrifice. To label cultured dermises,NMRI E13.5 dermises cultured with FGF20- or FGF9-loaded beads for 18 hr as described above. During the last 2 hr, 10 µM EdU (Life Technologies, Eugene, OR) was introduced in the medium. The samples were then fixed with 4% PFA and EdU detection was performed with Click-iT kit (Life Technologies, Eugene, OR) according to manufacturer’s protocol. Briefly, samples were permeabilized with 3% BSA, 0.5% Triton X-100 (MP Biomedicals, Solon, OH) for 1 hr, stained with Click-iT reaction cocktail containing Alexa488-azide for 2 hr protected from light, washed thoroughly for 2 hr with PBS and mounted in Vectashield and imaged using Lumar.V12 stereomicroscope with 1.2x objective and AxioCam ICc camera (all Zeiss).