Parameters included in systematic evaluation of staining conditions for CD31, CD8 and FOXP3 in osteosarcoma samples.

<p>Systematic evaluation of more than 200 different antigen retrieval conditions, different blocking conditions, detection systems and chromogens allowed identification of optimal staining conditions for formalin-fixed and paraffin-embedded osteosarcoma samples.</p><p>* In steps of 3–5°C;</p><p>** in steps of 5 min;</p><p>*** in concentrations of 0.1%–10%, adjusted temperatures and various times and combinations.</p

Correlation of common vessel quantification methods in osteosarcoma.

<p>Correlation of vessel quantification derived by total CD31-immunoreactive area and micro vessel density (A), respectively total CD31- immunoreactive area and Chalkley count (B) within 120 predefined spots with 0.26 mm² area/spot in 20 representative osteosarcoma sample; r indicates Pearson correlation coefficient.</p

Effect of heterogeneous vessel distribution on vessel quantification in osteosarcoma.

<p>Representative whole-slide scans of formalin-fixed and paraffin-embedded osteosarcoma samples with homogeneously scattered (A,C,E) and hot spot distributed intratumor vascularization (B,D,F) Quantification of tumor vascularization was either performed by hot spot analysis within three circular hot spots with 0.26 mm² area/hot spot (C and D) or whole-slide analysis of CD31-immunoreactive area (E and F). CD31 immunoreactivity is shown in red. By digital image analysis detected CD31-immunoreactive area is annotated in green (automated mark-up image). Indicated values represent the percentage of immunoreactive area within the analyzed regions (three hot spots in C and D, whole slide in E and F). Inserts show a 5-fold (C), respectively 8-fold (D) magnification of indicated regions. Sections were counterstained by hematoxylin.</p

Correlation of hot spot analysis and whole slide analysis of immunovascular markers in osteosarcoma.

<p>(A) Correlation between hot spot and whole slide analyses of CD31-immunoreactive area (in %) of 20 representative osteosarcoma samples. Circles filled in red represent the two specimens shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090727#pone-0090727-g003" target="_blank">Figure 3</a>. Pearson correlation coefficient is indicated by r, n.s. =  not significant. (B) Correlation between hot spot and whole slide analyses of FOXP3 cell density (cells per 0.1 mm²) of 20 representative osteosarcoma samples. Pearson correlation coefficient is indicated by r and significance by p. (C) Representative osteosarcoma sample with homogeneously scattered distribution of FOXP3 immunoreactive cells. Immunoreactive cells show red nuclear staining. Section was counterstained by hematoxylin. Insert shows 2.2 fold magnification of indicated area. (D) Representative osteosarcoma sample with homogeneous hot spot distribution of CD8 immunoreactive cells. Immunoreactive cells show red cell surface staining. Section was counterstained by hematoxylin. Insert shows 2.2 fold magnification of indicated area.</p

Effects of different staining conditions on the quantification of immunovascular markers in osteosarcoma.

<p>(A) Formalin-fixed and paraffin-embedded osteosarcoma sample after CD31 staining with standard heat induced epitope retrieval at 98°C and (B) with optimized enzymatic epitope retrieval. CD31- immunoreactive cells show red cell surface staining. Section was counterstained by hematoxylin. Insert shows 2-fold magnification of indicated area. (C) Formalin-fixed and paraffin-embedded osteosarcoma sample after FOXP3 staining with standard heat induced epitope retrieval at 98°C and (D) with optimized epitope retrieval at 127°C. FOXP3 immunoreactive cells (arrows) show red nuclear staining. Section was counterstained with hematoxylin. Insert shows 3.8 fold magnification of indicated area. (E) Percentage of CD31 immunoreactive area was assessed by computer-assisted whole-slide quantification after heat induced epitope retrieval (HIER) at 98°C, HIER at 127° and enzymatic epitope retrieval (EER) with Hyaluronidase and Pronase in five representative osteosarcoma samples (OS). Error bars indicate interobserver variability. (F) Only seven out of the 20 tested osteosarcoma samples showed FOXP3 immunoreactive cells after standard heat induced epitope retrieval at 98°C (not shown). Density of FOXP3-immunoreactive cells (numbers/0.1 mm²) was determined in these seven sections by whole-slide quantification after HIER at 98°C and for HIER at 127°C. Error bars indicates interobserver variability. OS = osteosarcoma sample. (G) Percentage of evaluable slides after standard and optimized immunohistochemical staining for CD31 and FOXP3.</p