Dako lsab kit

Endoscopic (mucosal) biopsy specimens from BE patients and matched tumor and adjacent normal specimens from EC patients, archived as formalin-fixed, paraffin-embedded material, were used for immunohistochemical analysis. Sections were routinely stained with hematoxylin and eosin and evaluated histopathologically. Tissue specimens were processed in a standard fashion, as described elsewhere 22 (link). ENO1 was identified using an anti-ENO1 mouse monoclonal antibody at a concentration of 0.025 μg/ml (Abcam, Cambridge, UK). Immunostaining was with a peroxidase-based visualization DAKO LSAB^® kit, following the manufacturer's recommendations. Diaminobenzidine tetrahydrochloride was used as chromogen. Sections incubated with PBS instead of the anti-ENO1 antibody served as controls.

Four proteins, p53, p16, p27 and c-erbB2, which have been demonstrated to be independent prognostic factors for non-small cell lung cancer (NSCLC) (13 (link)–16 (link)), were selected for the differential diagnostic analysis of MPLC and IPM. IHC staining was performed using serial sections obtained from the same paraffin-embedded blocks. The specimens were stained with hematoxylin and eosin in order to confirm the histological diagnosis. IHC staining was performed using the streptavidin-biotin-peroxidase complex method. For the antigen retrieval, sections were briefly immersed in a citrate buffer (0.01 mol/l citric acid; pH 6.0) and then incubated for 25-min intervals at 100°C in a microwave oven. Next, the sections were incubated with a monoclonal mouse anti-p53 antibody (dilution, 1:100; sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), a polyclonal rabbit anti-p16 antibody (dilution, 1:200; ab54210, Abcam, Cambridge, MA, USA), a monoclonal mouse anti-p27 antibody (dilution, 1:250; ab32034 Abcam) and a monoclonal mouse anti-c-erbB2 antibody (dilution, 1:100; ab2428, Abcam) overnight in a cold room using a labeled streptavidin biotin kit (Dako LSAB kit; Dako, Carpinteria, CA, USA). The antibodies were diluted in phosphate-buffered saline containing 2% bovine serum albumin.

38 paraffin liver cancer tissue samples were obtained from patients with hepatocellular carcinoma who underwent surgery in Peking Union Medical College Hospital from 2009–2010. All the patients were positive for HBV infection and none of them were found to have distant metastases before surgery. Cancer stages were classified according to TNM. They all underwent 1–3 times of transcatheter hepatic arterial chemoembolization (TACE) and the chemotherapy drugs were 5-Fu, epirubicin, HydroxycamptotbecineInjection (HCPT). The basic condition of patients were list in Additional file 1: Table S5.
CADM1 expression was evaluated by immunohistochemistric staining. Briefly, after 5-um sections were deparaffinized, antigen retrieval was performed by use of heat-induced epitoperetrieval with 10 mM citrate buffer. Sections were incubated with a monoclonal antibody against CADM1 (Abcam, UK) at 1 : 250 dilution. The CADM1 antibody was detected using the avidin-biotin-peroxidase technique (DakoLSAB Kit, Dako). The expression levels of CADM1 were determined by a pathologist. The classification of “-, +, ++” was defined by the percentage of CADM1 positive cells at the level of <10%, 10-50%, and 51-100%, respectively.

Mismatch repair status was determined using PCR or immunohistochemistry (IHC). For the detection of microsatellite instability (MSI) using PCR, 2~5 sections of FFPE tissue specimens at a thickness of 6 μm were used to extract DNA. Primers targeting microsatellite markers BAT-25 and BAT-26 and dinucleotide repeats D5S346, D2S123, and D17S250 were used to amplify mononucleotide repeats. Tumors were scored as follows: (1) MSI-high if two or more markers of instability were present, (2) MSI-low if one marker of instability was present, and (3) microsatellite stable (MSS) if none of the markers for MSI were present. For samples with no matched normal tissue, IHC was used to determine mismatch repair status. FFPE tissue specimens were processed using the streptavidin-biotin method with a DAKO LSAB kit (DAKO, Carpinteria, CA). Tumors that yielded negative staining results for at least one of the mismatch repair proteins (MLH1, MSH2, MSH6, or PMS2) were classified as MSI, whereas all others were classified as MSS tumors.

Under the approval of the Institutional Review Board of Taipei Veterans General Hospital (2015-06-005AC), OS tumor samples were collected from the Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, from 1992–2011. The cohort study was followed to determine mortality until 2014. The tumor size, and the levels of ALP and LDH were collected from medical record under routine inspection. The tissue arrays collected from 57 OS patients were analyzed by immunohistochemistry for the expression of ERα and P53. Missing samples, patients who were lost to follow-up, and duplicate collections were excluded, and a total of 50 tumor spots from each subject were analyzed in this assay. The dewaxed and rehydrated tissue array sections were retrieved by 10 mM sodium citrate buffer (pH 6.0) with 0.05% Tween 20 (Sigma, St. Louis, MO, USA) in a 95 °C water bath for 20 min and blocked by 3% H₂O₂ (Sigma, St. Louis, MO, USA). The antibodies against human ERα (mouse IgG anti-human, 1:100; GeneTex, Irvine, CA, USA) and P53 (mouse IgG anti-human, 1:100; Cell Signaling) were applied to the specimens. The DAKO LSAB kit (Dako Cytomation, Santa Clara, CA, USA) was used for detection. The expression levels of ERα and P53 in the tissue array sections were identified by more than two double-blinded researchers.