Biebrich Scarlet

Cells in lrECM gel were smeared on slides, dried briefly, and fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining was performed as previous described [32 (link)]. Stained samples were imaged with a Nikon upright epifluorescence microscope or a confocal system comprised of an Olympus IX81 microscope.
Xenograft tumor sections were de-paraffined and hydrated from xylene, 100% ethanol, 95% ethanol, 85% ethanol and 70% ethanol to distilled water. For Masson’s trichrome staining, slides were re-fixed with Bouin’s solution at 60°C for 60 minutes. Slides were washed in running tap water for 5 minutes and stained in Weigert’s working hematoxyin for 10 minutes. Then they were washed in running tap water for 5 minutes and stained in Biebrich scarlet-acid fuchsin solution for 5 minutes. Slides were rinsed in distilled water and differentiated in phosphomolybdic-phosphotungstic acid solution for 10 minutes, transferred to aniline blue solution and stain for 5 minutes. Slides were rinsed in distilled water and images were taken with a Nikon microscope. The percentage of collagen was quantified by calculating the ratio of blue staining (collagen) area in the total area of the tumor section using Imagescope analysis software [33 (link)].

PAS staining and Masson’s trichrome staining were performed as described in detail previously50 (link)51 (link). Cross-sections of the kidneys were fixed in 10% buffered formaldehyde solution and embedded in paraffin. The sections (5 μm) were obtained and used for histological and immunohistochemical analyses. The sections were stained with PAS method. The slides were examined under light microscopy by a pathologist in a blinded manner. Histological findings were graded according to the modified Banff classification criteria in 20 randomly selected non-overlapping fields per PAS staining.
For renal fibrosis, Masson’s trichrome staining was used to evaluate the extent of renal fibrosis according to the standard Masson’s trichrome protocol. Briefly, kidney tissue sections were successively immersed into Weigert’s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. To quantify the renal fibrosis, the blue pixel contents of the images were photographed with the same microscope and magnification times. Ten different views in each group were selected to detect the values of the integral optical density and the total area and the expression intensity was calculated as the percentage of the integral optical density to the total area which was performed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.). The measurements for each of the samples for PAS staining and Masson’s trichrome staining were replicated 3 times.

All the kidney tissues were excised, fixed in 4% formaldehyde and embedded in paraffin. Kidney tissue sections (5 μm) were obtained and used for histological and immunohistochemical analyses. H&E staining was performed according to the standard H&E protocol. The injury score was acquired by modified Banff classification criteria in ten randomly selected non-overlapping fields per rat H&E stained kidney tissues59 (link). All specimens were blindly evaluated by one nephrologist.
For renal fibrosis, Masson’s trichrome staining from the each group was used to evaluate the extent of renal fibrosis according to the standard Masson’s trichrome protocol. Briefly, kidney tissue sections were successively immersed into Weigert’s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. To quantify the renal fibrosis, the blue pixel contents of the images were photographed with the same microscope and magnification times. Ten different views in each group were selected to detect the values of the integral optical density and the total area and the expression intensity was calculated as the percentage of the integral optical density to the total area which was performed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

The toxicity of repeated and high dose of CNPs treatment was assessed by histological and hematological analyses. Briefly, Cy5.5-CNPs (10, 22.5 or 90 mg/kg) were intravenously injected into BALB/c mice with single- or multi-dosage (three times). On day 7 after treatments, major organs (liver, lung, spleen, kidney, brain and heart) were collected from mice, and structural abnormalities in organ tissues were assessed by staining with H&E. In the case of hematological analyses, blood samples were collected from the mice on day 7 and centrifuged at 2200 rpm to obtain plasma. The following factors in blood samples were measured; alanine aminotransferase (ALT), blood urea nitrogen (BUN), alkaline phosphatase (ALP), aspartate Aminotransferase (AST), creatine kinase (CK) and troponin I. The cardiotoxicity by Cy5.5-CNPs was further analyzed after multiple-dosage. The heart tissues were collected from mice after treatment with 10, 22.5 or 90 mg/kg of Cy5.5-CNPs three times. The accumulation of Cy5.5-CNPs in heart tissues was observed using a Leica TCS SP8 confocal laser-scanning microscope. Collagen fiber in heart tissues were stained with Masson's trichrome. Briefly, heart tissues were incubated in Bouin's fixative for 30 min at 56 °C, and the nuclei were co-stained with Weigert's iron hematoxylin. Then, cytoplasm was stained with Biebrich scarlet-acid fuchsin, and then differentiated in phosphomolybdic–phosphotungstic acid. The collagen matrix in heart tissues was stained with aniline blue solution. The collagen in heart tissues were quantitatively analyzed using an Image Pro software, and collagen contents were presented in proportion to the total area of heart tissues.

Through the modified and adapted inflammatory score grading scale, the following scores were assessed for microscopic anatomopathological analysis (semi-quantitative): histological characteristics related to the inflammatory process (presence of leukocyte infiltration/inflammatory exudate and active hyperemia) and characteristics related to tissue proliferation (presence of granulation tissue and re-epithelialization).
Hematoxylin–eosin (HE) was used for semi-quantitative analysis of inflammatory parameters (inflammatory exudate and active hyperemia) and proliferative parameters (granulation tissue and re-epithelialization) through the modified and adapted inflammatory/proliferative score scales and with Masson’s Trichrome (MT) for quantitative analysis of total collagen [134 (link)]. Briefly, for the HE technique, the slides were stained with hematoxylin for 5 min, washed in running water, stained with eosin for 3 min, and, finally, washed in running water. For the MT technique, the slides were kept for 1 h in Bouin’s solution in the oven, cooled, washed in running water until completely clear, washed in distilled water 3 times, and treated with Biebrich’s Scarlet solution for 3 min. Again, they were washed in running distilled water, kept in phosphotungstic acid for 8 min, washed in running distilled water, kept in aniline blue for 3 min, and, finally, washed in running water once again.
Each of the parameters (inflammatory exudate, active hyperemia, granulation, and re-epithelialization) were stratified as: 0 = absent; 1 = mild; 2 = moderate; and 3 = intense, according to the changes found using HE (Table 2 and Table 3) [134 (link)]. Each parameter in each experimental group was analyzed separately by an experienced collaborating pathologist who was unaware of the experimental groups to which the animals belonged. For this purpose, the slides (HE) were analyzed in 3 different fields, and the value adopted for each parameter analyzed for each animal in the group was the mean value found after reading three different fields of the lesion area. The final value assigned to each sample analyzed in each experimental group was the mean value obtained by adding the values of each parameter [134 (link)]. The 500× final magnification data were used for the analysis.
The total collagen content was analyzed by quantitative reading on the slides in three different fields. The computer-assisted image analysis program was used. The selected image was captured by a video camera previously coupled to an optical microscope (Eclipse DS50—Nikon Inc., Osaka, Japan) and then analyzed by the program NIS-Elements (Nikon Inc., Japan) [134 (link)]. By means of color histograms, the software determines the color intensity of each area selected for measurement, transforming the chosen color into a numerical expression for each selected field of view. Using the color histogram in the RGB (red, green, blue) system, the blue color was selected, the intensity of which was captured by the number of pixels containing the color and then converted into a numerical value. The final value considered for each measured field of each sample was represented by the average of the values found after the evaluation of three different fields [134 (link)]. For both analyses, a final magnification of 500× was used.