Hematoxylin qs

Tissue microarray covering tissue sections of oral cavity carcinoma progression, OR802 (10 cancer adjacent tissues or normal tissues, 7 hyperplasia epithelia, 8 benign tumors, and 45 oral carcinoma) were purchased from US BIOMAX. Immunohistochemistry was performed as previously described [35 (link)]. Briefly, all paraffin sections were dewaxed and rehydration followed by heating in a 0.01 M citrate buffer for 20 min. Subsequently, anti-LDOC1 (1:2000 dilution, LifeSpan, Newton, MA, USA) was incubated overnight at room temperature. The secondary biotinylated antibody and the streptavidin–peroxidase conjugate (BioGenex, Netherlands) were then incubated on the sections for each 20 min. The sections were stained with diaminobenzidine (DakoCytomation) for detection and stained with hematoxylin QS for counterstaining (Vector, Burlingame). Normal serum and phosphate buffer instead of specific antibodies was used as negative controls. Immunoreactivity was scored by the staining intensity using MetaMorph software (Molecular Devices). Staining intensities < 0.15 was described to be low LDOC1 protein expression, staining intensities > or = 0.15 was described to be high LDOC1 protein expression.

The lungs of the F344 rats were embedded in paraffin wax at the Department of Pathology, Nanjing Medical University (Jiangsu, China), using routine methods. Sections (5 μm) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. Some sections were treated with H&E staining and examined by light microscopy to determine the pathological features of the lung tissues.
For the remaining sections, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in Tris-buffered saline. Some of these tissue sections were then incubated with rabbit polyclonal anti-PA primary antibody (Pierce, USA), followed by the EnVision HRP complex (DAKO, Carpinteria, CA). They were then counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA). The results were analyzed according to the IHC score (IHS) as described previously36 (link). Briefly, the IHS was determined by evaluation of both staining density and intensity. Multiplication of the intensity and percentage scores yielded the final IHS. Samples with IHS ≤3 were considered weakly positive, while those with IHS ≥6 were considered strongly positive. The IHC results were evaluated by two independent investigators blinded to the rat groups. In cases of conflict, a pathologist reviewed the cases, and a consensus was reached.

RNAscope in situ hybridization was carried out on formalin-fixed paraffin-embedded human joint replacement tissues sections of OA and RA synovium provided by the Royal Orthopaedic Hospital (ethical approval no. 07/H1204/191). Sections were deparaffinized in xylene, followed by 100% ethanol. Detection of human C15orf48 transcript was performed using an RNAscope 2.5 HD manual assay red kit reagents and methods according to the manufacturer (ACD Bio-Techne). Following the last wash for the RNAscope protocol, slides were washed in distilled water for 5 min and then blocked with Bloxall (Vector Laboratories) for 10 min, followed by incubation with 10% normal horse serum in tris buffer for 10 min. Immunohistochemistry was performed by incubating sections overnight at 4°C with biotin-conjugated mouse anti-human CD68 (Novus, NBP2-34661B), followed by streptavidin-HRP (Thermo Fisher Scientific) and ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories). Hematoxylin QS (Vector Laboratories) was used for counterstain. Images were obtained using the Zeiss Axio Scan and analyzed in Zen Blue (both Zeiss).

A LMD7000 (Leica, Buffalo Grove, IL, USA) system was used for LCM. Liver tissue embedded in OCT was cut with a cryostat at 12–14 μM thickness and then mounted onto polyethylene naphthalate (PEN) membrane glass slides (MDS Analytical Technologies, Sunnyvale, CA, USA) and subsequently fixed in 100% ethanol at 4C for 15 min. After air-drying, the sections were stained with Hematoxylin QS (Vector Laboratory, Burlingame, CA, USA) for 30 s. Thereafter, the slide was dipped in 95% ethanol. The air-dried slides were subjected to LCM at the parameters of power: 30, aperture: 20, speed: 10, specimen balance: 25, head current: 100, pulse frequency: 800. Multiple lobules from each subject were microdissected, and the samples from each zone were pooled in RLT buffer (Qiagen, Valencia, CA, USA) and stored at −80C.

Paraffin-embedded livers were cut into 4 μm sections and mounted onto positively charged slides (VWR, Radnor, PA, USA). Slides were deparaffinized and stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA, USA) for 1 min followed by staining for 1 min with eosin Y (Amresco, Solon, OH, USA) and rinsed in 95% ethanol. The slides were then dipped into 100% ethanol and subsequently through two xylene washes. Coverslips were mounted onto the slides using CytoSeal XYL mounting media (ThermoFisher). The slides were viewed and imaged using an Olympus BX40 microscope with an Olympus DP25 imaging system (Olympus, Center Valley, PA, USA). Serum alanine aminotransferase (ALT) concentrations were measured using a commercially available kit from Sigma-Aldrich with all assays and subsequent analyses performed according to the instructions provided by the manufacturer.