Axiophot optical microscope

The statistical analysis was carried out using the program GraphPad Prism^® 6.0. The Mann–Whitney U and Pearson χ² tests were studied in this work. The obtained data are expressed as the median with interquartile range (IQR) and significance was established at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Five sections and ten fields per section were randomly analyzed for each patient classified in their established groups. Patients were considered positive when the marked average area in the analysed sample was ≥5% of the total, following the pathology protocol. The slides were examined under a Zeiss Axiophot optical microscope (Carl Zeiss, Germany).

For the statistical analysis, GraphPad Prism® 6.0 was used to conduct a Whitney U test. Data are expressed as the median with interquartile range (IQR). The significance was established at a value of p <0,05 (*), p <0,01 (**), p <0,001 (***). To quantify the immunopositive cells, 5 counts were randomly applied to tissue sections, excluding cells not entering the designated demarcation lines. The percentage of positive cells was calculated by the following formula: % Positive cells = No. Cells+/(No. Cells- +No. Cells-) × 100. Patients were described as positive when theme an of the test sample scored for each subject was greater than or equal to 5% over the total , following the anatomo-pathological criteria stated by Cristóbal et al. 22 (link) this procedure is a minimal modification of the immunoreactive score (ISR score). Immunostaining in the tissue was assessed by two independent histologists (M.AO. and M.A.S.) blinded to the outcome measure. The cuts were studied under a Zeiss Axiophot optical microscope (Carl Zeiss, Germany).

Five different sections and ten fields were randomly examined for each patient. Immunohistochemical expression was classified as positive(+) when the mean stained area in the studied sample was equal or superior to 5% of the total, following the immunoreactive score (IRS) [37 (link)]. This method allowed us to classify immunostaining using the following scale: 0–1, minimum staining (≤25%); 2, moderate staining (25–65%) and 3–4, strong staining (≥65–100%). Two independent histologists evaluated each sample. A Zeiss Axiophot optical microscope (Carl Zeiss, Oberkochen, Germany) was used for histological determination.
For statistical processing, the GraphPad Prism® v6.0 (GraphPad, Inc., San Diego, CA, USA) program was employed. The Mann–Whitney U test was performed to compare both groups, and the results are expressed as the median ± SD. Significant values were established as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

For statistical analysis, the GraphPad Prism® 6.0 program was used to apply the Mann–Whitney U test. The data are expressed as the mean ± standard deviation of the population. Significance was established at ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.001. In the case that the study variables were not qualitative, a Pearson chi-square test or Fisher's exact test was used when applicable. In the case of inequality, possible confounding factors were defined for which the final analysis of the main efficacy variable was adjusted.
For each of the patients in the established groups, five sections and 10 fields per section were randomly selected and examined. The patients were described as positive when the average of the analysis of the labeled sample for each study subject was greater than or equal to 5% of the total, following the anatomopathological protocol of Ortega et al. [24 (link)]. Infiltrated cells were counted under a microscope (1000x) in 10 aleatory areas of 0.5 mm² per patient. All values are expressed as means ± SE. Sample observation was carried out using a Zeiss Axiophot optical microscope (Carl Zeiss, Germany) equipped with an AxioCam HRc digital camera (Carl Zeiss, Germany).

A total of 5 different sections and 10 areas of view were randomly examined for every patient of CVD and HC groups. A patient was reported as positive when the stained mean area in the studied sample was ≥5% of the total, following the immunoreactive score (IRS) as established in previous studies [22 (link)]. Immunostaining was evaluated by two independent histologists, and then each sample was scored using the following scale: 0–1, minimum staining (≤25%); 2, moderate staining (25–65%); and 3–4, strong staining (≥65–100%). A Zeiss Axiophot optical microscope (Carl Zeiss, Oberkochen, Germany) was used for the histological determination.
For the treatment of statistical data, GraphPad Prism^® v6.0 (GraphPad, Inc., San Diego, CA, USA) program was utilized. Mann–Whitney U test allow the comparison between both groups, being expressed as the median ± SEM. Significant values were established as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

After fixation and decalcification of the samples by Osteosoft^®, the inclusion and cutting process was performed. Different histological techniques were performed: hematoxylin–eosin (HE) for the staining of bone and cartilage tissue, Gram staining for the staining of bacteria, and immunohistochemistry. The monoclonal antibody used to detect macrophages in rabbits was RAM-11 (DAKO ref. M633 dilution 1:50). Immunohistochemical detection of the antigen of interest was performed using the avidin–biotin complex method, using alkaline phosphatase as a tracer and Fast Red as a revealing solution. In all cases, the same biological material without primary antibody was used as a negative control. Staining was visualized under a Zeiss Axiophot optical microscope (Carl Zeiss, Oberkochen, Germany).

For the statistical analysis, GraphPad Prism® 6.0 was used. The Mann–Whitney U test was applied, and the Pearson χ² test was used. The data are expressed as the median with interquartile range (IQR). Significance was established at values of p < 0.05 (^∗), p < 0.01 (^∗∗), and p < 0.001 (^∗∗∗). For each of the patients in the established groups, 5 sections and 10 fields per section were randomly selected. Patients were described as positive when the marked average area in the analyzed sample was greater than or equal to 5% of the total, according to the IRS score following the anatomical protocol of Cristóbal et al. [21 (link)]. The preparations were examined under a Zeiss Axiophot optical microscope (Carl Zeiss, Germany).