Bond 3 automated system

Chordoma tissues from the 187 chordoma patients were fixed with formalin and embedded in paraffin for following TMA construction. After assayed by TMA using the Tissue Array MiniCore (ALPHELYS, Plaisir), the most representative parts of the tissue were selected to build the TMA. Then TMAs were cut into 4μm thick sections for further staining with Leica RM 2135 Rotary Microtome (Rankin, Wetzlar, Germany). Leica BOND III automated system was used to perform Immunohistochemical staining with primary antibody (anti-PALB2 antibody, ab-202970, abcam, 1:500) and Bond Polymer Refine Detection (Leica Biosystems, DS9800) including secondary antibody. Leica Aperio AT2 scanner was used to scan TMA images at 400× magnification. Three pathologists examined the slides independently. Then, we analyzed the images by Leica Aperio ImageScope software (version 12.3). The H-score was used to evaluate staining intensity. H-Score (12 (link)) = 1 × (percent of light staining cells) + 2 × (percent of moderate staining cells) + 3 × (percent of strong staining cells).

IHC staining was performed using a Leica BOND III automated system. Anti‐FAP antibody (ab207178, 1:400) was used for the IHC staining of tumor tissues from patients with glioma. Immunostained slides of TMA sections were scanned using a Leica Aperio AT2 scanner (at 400× magnification) and analyzed using a Leica Aperio ImageScope v12.3. The automated algorithm scored the staining of each tissue core as negative (0), weak (1+), moderate (2+), or strong (3+) according to the scoring criteria threshold. The algorithms also determined the percentage of positive staining. Then, the H‐score of each case was established using the formula H‐score = 1 × (percentage of 1+ cores) + 2 × (percentage of 2+ cores) + 3 × (percentage of 3+ cores). Thus, the H‐score ranged from 0 to 300.¹³ FAP (ab207178), Ki‐67 (ab15580), E‐cad (Cell Signaling Technology, #14472), and N‐cad (Cell Signaling Technology, #13116) antibodies were used for the IHC staining of subcutaneous tumors from BALB/c athymic nude mice.

SKOV3-ip^LucshSC or SKOV3-ip^LucshJLP cells (5 × 10⁶ in 0.2 mL PBS) were intraperitoneally injected into the lower right quadrant of 1.5-month old female nu/nu nude mice (Charles River, Wilmington, MA). The progression of injected cells was monitored by in vivo bioluminescence on an IVIS Spectrum imaging system (PerkinElmer, Waltham, MA). Intraperitoneal injection of D-Luciferin (PerkinElmer) was administered to the isofluorane-anesthetized mice at 150 mg/kg of body weight. After 10 minutes, the mice were re-anesthetized and the bioluminescent images were taken at both ventral and dorsal positions. The radiance of the bioluminescence was quantified as photons per second. After euthanasia, the xenograft tumors were excised, fixed in 10% formalin, and used to create the tumor tissue microarray (TMA). TMA was constructed at the Cancer Tissue Pathology Core of the Stephenson Cancer Center. IHC staining was carried out as described above on a BOND-III automated system (Leica, Buffalo Grove, IL). Intensity of strong positive (Isp) per square micrometer of the IHC image was quantified using Spectrum software (Aperio, Vista, CA).

Two 2.0-mm diameter samples were removed then transferred to a recipient paraffin block to construct TMAs using a Tissue Array MiniCore 3 (ALPHELYS, Plaisir, France). Samples (4-µm thick) were obtained from each TMA with Leica Rotary Microtome RM2135 (Wetzlar, Germany). Immunohistochemical staining was performed on Leica BOND III automated system. All samples were pretreated in a 65 °C oven for 1.5 h. Staining was performed with dewaxing and epitope retrieval. Samples were blocked with peroxide solution for 7 min. Selected antibodies (shown in Additional file 1: Table S1) were used to incubate samples, followed by post-primary for 8 min, polymer for 8 min, diaminobenzidine (DAB) for 5 min, and hematoxylin counterstain for 2 min. Finally, the samples were dehydrated, cleared, then fixed with neutral resins. Stained samples were scanned with Leica Aperio AT2 scanner (400× magnification) and analysed by two different pathologists. Samples were scored as negative (0+), weak (1+), moderate (2+), and strong (3+) signal. The percentage of positivity was also calculated by two pathologists. The histological scores (H-Score) was calculated using following formula: H-Score = 0 × (percentage of negative) + 1 × (percentage of weak) + 2 × (percentage of moderate) + 3 × (percentage of strong). Thus, the H-Score ranges from 0 to 300 [5 (link)].