Labophot

Heart tissues were fixed in 10% buffered formaldehyde and then embedded in paraffin. Cross sections (4μm thick) of the LV were stained with hematoxylin and eosin stain (H&E) and observed under a light microscope (Nikon Labophot, Japan). Cardiomyocyte diameters were measured by two investigators who were blind to the animal groups. In each sample, approximately 50 cardiomyocytes from the LV were examined, and the cardiomyocyte diameters were measured. The mean value of 50 measurements indicated one sample [1 (link)].

Bacterial suspensions (1 ml) were mixed with 1.5 μl of Syto 9 (absorption/emission 485/498 nm) and 1.5 μl propidium iodide (absorption/emission 535/617 nm) from LIVE/DEAD^® BacLight^TM Bacterial Viability Kit (Life Technologies) and incubated in the dark for 20 min at room temperature. A 5 μl aliquot was applied to the center of a clean glass microscope slide. An 18 mm² coverslip was placed over the suspension. The slides were examined over 10 min at a magnification of 1,250 (100×, plan Apo) with an epifluorescence microscope (Labophot; Nikon, Tokyo, Japan) and images captured from 10 independent fields using a vertical mounted digital camera.

Two groups were evaluated: hydrogel made by in natura porcine skin (control), and decellularized porcine skin, both lyophilized, to evaluate by hematoxylin and eosin (HE) staining the presence of cells in the hydrogel after the protocol for decellularization. To compare the collagen fibers distribution and morphology between the two samples, it was used picrosirius staining. In both protocols, the hydrogel was spread on a glass blade and fixed with formaldehyde 4% for 24 hours.
For typical HE staining, the fixed hydrogel blades were immersed into hematoxylin solution for 3-5 minutes and washed with dH₂O three times. Then, they were immersed into eosin solution for maximum 3 minutes and washed with different concentrations of ethanol (70, 80, 90, 95, and 100%) and xylene. For picrosirius staining, after dH₂O wash, the slide was immersed into Sirius red working solution for 1 hour before the steps of alcoholic dehydration. HE analysis was quantitative, and the cell nuclei were counted in ten distinct fields of three glass blades of each group. The microscope used was Labophot (Nikon, United States of America).

We prepared embryonic cuticles according to (Wieschaus and Nüsslein-Volhard, 1986 ). Embryos were collected on apple juice agar plates with yeast, aligned on a fresh apple juice agar plate without yeast and incubated at 25°C for 48 h to allow embryos to develop fully and viable embryos to hatch. All unhatched embryos were collected in 0.1% Triton X-100 and dechorionated in 50% bleach for 5 min. They were then washed three times with 0. 1% Triton X-100 and transferred to glass slides, where all the liquid was removed, mounted in 1:1 Hoyer's medium:lactic acid, and incubated at 60°C for 24–48 h. They were then stored at room temperature. Images were taken using a Nikon Labophot with a 10× Phase 2 lens, and captured on an iPhone, and placed into categories based on morphological criteria.

Hematoxylin and eosin (H&E) were used to stain the prepared liver sections, and then they were observed under a light microscope in a blind manner (Nikon Labophot, Japan) with the help of an expert pathologist. The Ishak fibrosis score and the modified Knodell histology activity index (HAI) and Ishak fibrosis score were utilized to score the biopsy specimens (25 (link), 26 (link)).

Mean Feret’s diameter along with mean roundness and mean elongation ratio was determined using an optical microscope (Nikon Labophot, Nikon, Japan) connected to a camera (Panasonic camera WVCL310, Panasonic, Japan). The image of a pellet was captured and processed using particle size analysis software V1999 (designed in-house at King’s College London). This was repeated so that 100 pellets were analysed per formulation in order to obtain Feret’s diameter and also shape factors such as roundness and elongation ratio. Roundness and elongation ratio were calculated using Eqs. 1 and 2 respectively [24 ]. Only physical mixture pellet and two best-optimised formulations underwent stereoscopic analysis.
$$\text{Roundness}={\left(\text{perimeter}\right)}^2/\left(4\mathit{\pi A}\right)$$
$$\text{Elongation ratio}=\text{Maximum Feret diameter}/\text{Minimum Feret diameter}$$

Intensity and frequency of root colonization by AMF were measured according to⁶⁶ after roots staining with Trypan blue following the method described by Phillips & Hayman^{67 (link)}. Stained root fragments were observed with a Nikon Labophot trinocular microscope. For each fragment, a score between zero and five was assigned according to the estimated proportion of root cortex colonized by AMF⁶⁶ .
Frequency and intensity of root colonization were then computed using the following formulas: $$\text{Frequency}\left(\text{expressed in}\%\right):\text{F}=\text{n}/\text{N}\times 100$$ where n is the number of fragments showing mycorrhizae and N, the number of observed fragments $$\text{Intensity}\left(\text{expressed in}\%\right):\text{I}=\left(\text{95n5}+70\text{n4}+30\text{n3}+\text{5n2}+\text{n1}\right)/\text{N}$$ where n1, n2, n3, n4, n5 are the number of fragments scored respectively from 1 to 5 and N, the number of fragments observed.