Axiophot light microscope

For statistical analyses, GraphPad Prism^® 5.1 software was used with Mann‐Whitney U test. The data are presented as the median with interquartile range (IQR). Significance was set at P < .05 (*) and P < .005 (**). For each patient in the established groups, five sections and 10 fields per section were randomly selected and examined. Patients were characterized as positive when the average marked area in the sample analysed was greater than or equal to 5% of the total area, following the anatomical‐pathological protocol of Cristóbal et al³⁰ (link) In each sample, immunohistochemical staining was scored using the following scale: 0‐1, minimum staining (0%‐25%); 2, moderate staining (25%‐65%); 3, strong staining (65%‐100%). This procedure is a minimal modification of the immunoreactive score (ISR score).³⁰ (link) The preparations were examined under a Zeiss Axiophot light microscope (Carl Zeiss) equipped with an AxioCam HRc digital camera (Carl Zeiss).

The expression patterns of two known piRNA pathway genes from chicken, CIWI and CILI, were examined using qPCR and in situ hybridization. For qPCR, cDNA samples from PGCs, stage X blastoderms, GSCs, and CEFs were amplified using the appropriate CIWI and CILI primers (Additional file
10: Table S7), as described above. For in situ hybridization, cDNA from PGCs was amplified using primers targeting CIWI (F: 5′-CCT GAT GGT GTA GGA GAT GGA; R: 5′-CAA GGA AAG CCA GTT TAT GGG) [GenBank: NM_001098852] and CILI (F: 5′-TGA GCC CCG ACA TCC ACA G; R: 5′-TTC TTG GGC AGG CAG TGG TT) [GenBank: JN248386]. The CIWI and CILI PCR products were cloned into the pGEM-T plasmid vector (Promega) and transformed into E. coli strain DH5α. After we verified the cloned sequence, the recombinant plasmid containing CIWI and CILI was amplified using T7- and SP6-specific primers and was subjected to cRNA probe preparation using a digoxigenin RNA labeling kit (Roche Diagnostics). Localization of CIWI and CILI during limited stages of germ cell development (E13.5, E15.5, E17.5, 1 day, and 24 weeks) in male and female chickens was examined as described previously
[53 (link)]. The mRNA signal was visualized as a brown color, and images were captured under a Zeiss Axiophot light microscope (Carl Zeiss).

After animal euthanasia at 14 days postsurgery, specimens were collected from the implants and omental adhesions to the mesh. Non-injured and adhesion-free omental tissue was also collected from each animal. For light microscopy, the specimens were fixed in F13 solution (60% ethanol, 20% methanol, 7% polyethylene glycol, and 13% distilled water), embedded in paraffin, cut into 5-μm-thick sections and subjected to hematoxylin-eosin, Masson’s trichrome (Goldner-Gabe) and Picrosirius red staining. These sections were then examined with a Zeiss Axiophot light microscope (Carl Zeiss, Oberkochen, Germany). Picrosirius red staining was also observed under polarized light, which allowed observation of collagen levels and different collagen fibers orientations.
Specimens (approximately 1 cm²) consisting of the mesh and the underlying abdominal wall were fixed in 3% glutaraldehyde and processed as previously described in the scanning electron microscopy section.

Leaf samples (n = 5) were chemically fixed in 4% (wt/vol) paraformaldehyde and 2.5% (wt/vol) glutaraldehyde in 0.05M Sorenson's phosphate buffer pH 7.2. Samples were washed three times in 0.05M Sorenson's phosphate buffer, dehydrated in a graded ethanol series and infiltrated in increasing concentrations of LR White Resin (medium grade Agar, AGR1281). Samples were polymerised at 60°C for 16–20 hr in a nitrogen rich environment and semi‐thin sections (1 μm) cut using a Leica rotary microtome RM 2265 (Leica Biosystems, Milton Keynes, UK). Sections were collected on drops of distilled water on glass slides coated with poly‐l‐lysine and dried on a hot plate at 60°C. The sections were stained with 1% (wt/vol) Toluidine blue in 1% (wt/vol) sodium tetraborate buffer pH 9 for 1 min and rinsed in distilled water for 1 min. Toluidine blue was used to highlight general histological features. Images of tissues of different genotypes were acquired with a Zeiss Axiophot light microscope (Carl Zeiss Ltd., Cambridge, UK) equipped with a Q‐Imaging Retiga Exi Fast 1394 monochrome camera (QImaging, Surrey, BC, Canada) and Metamorph imaging software version 7.8.13 (Molecular Devices, LLC, Sunnyvale, CA).

Histological analysis of the human skin was performed in both groups after wound closure by two independent histologists. Samples of skin were obtained from the two groups, and they were placed in 10% buffered formaldehyde (for histopathological studies) and Bouin (for immunohistochemical studies). Then the samples were dehydrated and embedded in paraffin. Tissue sections (5 mm thick; 50 sections for each sample) passing through the central plane of each sample were stained with hematoxylin-eosin and Masson’s trichrome for morphological assessment. Tissue sections were examined under a Zeiss Axiophot light microscope (Carl Zeiss, Oberkochen, Germany). The thickness of the healed human skin was measured in micrometers with Axiovision Release 4.6.3 software (Carl Zeiss, Oberkochen, Germany). The total thickness of the skin was defined as the distance between the stratum corneum and the boundary between human dermis–host mouse tissue.

Tissue sections were visualized using a Zeiss Axiophot light microscope (Carl Zeiss, Oberkochen, Germany) equipped with an AxioCam HRc digital camera (Carl Zeiss, Oberkochen, Germany). The histological evaluation was carried out according to the intensity of the expression of the immunohistochemical staining with the IRS-Score method [28 (link)]. Therefore, histological samples from patients diagnosed with pancreatic cancer were classified as negative expression (0), low/moderate (1) and high (3). For every group of subjects, seven randomly selected microscopy fields were examined in each of the five sections. Positive individuals were classified when the mean proportion of the labeled sample was superior or equal to 5% of the total sample. This was achieved by calculating the total percentage of marked tissue in the different microscopy fields to obtain an average of the study sample as described in previous works [29 (link)]. The quantification and observation of the different samples were performed separately by two independent researchers.