Flex ihc microscope slides

The formalin-fixed, paraffin-embedded tissues were cut into 3-µm sections and dried on Flex IHC microscope slides (Dako). The sections were deparaffinized with standard xylene and hydrated through graded alcohols into water. Antigen retrieval was performed using Envision Flex Target Retrieval solution, high pH (Dako). Staining was done at room temperature on an automatic staining workstation (Dako Autostainer Plus) with Nanog (D73G4) XP® Rabbit monoclonal antibody (Cell Signaling technology, Inc.) at 1:200 dilution using the Dako EnVision Flex + Visualization System (Dako Autostainer). Counterstaining with haematoxylin was the final step.
A semiquantitative scoring system based on staining intensity was applied, as previously reported^{5 (link)}. Immunostaining of the dysplastic areas was scored blinded to clinical data by two independent observers as negative (absence of staining, score 0), weak to moderate (some cytoplasmic staining in dysplastic areas, score 1), and strong protein expression (intense and homogeneous cytoplasmic staining in dysplastic areas, score 2), with a high level of inter-observer concordance (>95%). As in some cases nuclear staining was observed, the cases were also scored as positive/negative based on the presence of nuclear staining in dysplastic areas. Human seminoma was used as positive control, showing strong nuclear NANOG staining.

hUCESCs were cultured as described above. 3 × 10⁴ cells were seeded in slides, and fixed for 10 minutes in 96% ethanol, before processing for immunocytochemistry. Mouse tumors were immersion-fixed in 10% neutral buffered formalin for 24 hours and routinely embedded in paraffin. 4 μm thick sections were mounted on Flex IHC microscope slides (Dako, Glostrup, Denmark). The immunohistochemical (IHC) techniques were automatically performed in an AutostainerLink 48 (Dako). FLEX ready-to-use Dako primary antibodies to CK (clone AE1/AE3), E-cadherin (clone NCH-38), vimentin (clone V9), desmin (clone D33), actin (clone HHF35), smooth muscle actin (clone 1A4), β-catenin (clone beta-catenin-1) were employed. KLF4, OCT4, and Sox2 primary antibodies were obtained from Santa Cruz Biotechnology (Dallas, USA), Millipore, and Sigma-Aldrich, respectively. Epitope retrieval was performed in a PT Link (for 20 minutes at 97ºC) using EnVision FLEX target retrieval solution (pH 9). All antibodies were incubated for 20 minutes at RT. As detection system we used EnVision FLEX/HRP Dako (dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-mouse and anti-rabbit immunoglobulins) for 20 minutes. For E-cadherin, a mouse linker (Dako) was added. Quantitation of immunopositive cells for active caspase-3 expression was performed as previously described [57 (link)].

Full-faced formalin-fixed, paraffin-embedded tumor and non-tumor tissues (FFPE) were obtained from the Department of Pathology at Sahlgrenska University Hospital in accordance with the Declaration of Helsinki. Our study is not a clinical trial and the tumor specimen were used anonymously therefore, patient consent is not needed and the research on these tumors is approved by the Medical Faculty Research Ethics Committee, Gothenburg, Sweden (s164-02). In addition, the review board waived the need for written informed consent from the participants. All samples were obtained from patients undergoing surgical resection in Gothenburg, Sweden, between 1990 and 2006. FFPE sections, 4 µm thick, were applied onto positively charged slides (FLEX IHC microscope slides, Dako, Sweden) for immunostaining, in order to assess NLK protein expression in tumor as well as adjacent normal breast tissue.

The formalin-fixed, paraffin-embedded tissues were cut into 3-μm sections and dried on Flex IHC microscope slides (Dako). The sections were deparaffinized with standard xylene and hydrated through graded alcohols into water. Antigen retrieval was performed using Envision Flex Target Retrieval solution, high pH (Dako). Staining was done at room temperature on an automatic staining workstation (Dako Autostainer Plus) with rabbit polyclonal anti-HERG1A (NT) antibody (Enzo Life Sciences, Inc.) at 1:250 dilution using the Dako EnVision Flex + Visualization System (Dako Autostainer). Counterstaining with haematoxylin was the final step.
Since HERG1A staining showed a homogeneous distribution, a semiquantitative scoring system based on staining intensity was applied. Immunostaining was scored blinded to clinical data by two independent observers as negative (0), weakly (1+), moderately (2+) or strongly positive (3+). Scores ≥2 were considered as HERG1A-positive expression.

CK19 IHC was performed on representative sections of all the primary colon carcinomas to ensure reliable negative molecular CK19 mRNA results. A 2-μm section of each primary tumour was mounted on FLEX IHC microscope slides and pre-treated in PT-LINK (Dako, Glostrup, Denmark). Incubation for 20 min with the primary CK19 antibody (CK19 mouse monoclonal, clone RCK108; IR615 pre-diluted. Dako) was performed in the AutostainerLink 48 (Dako). Membranous staining with or without cytoplasm staining of ≥10 % of the tumour cells was defined as positive IHC in colon carcinomas (Fig. 1a, b).Fig. 1

a A primary high-grade mucinous carcinoma with signet ring cells. b Positive CK19 immunohistochemistry from the same case. c Amplification curve of CK19 mRNA from a positive LN, assessed through turbidity variation (Y-axis) over time (X-axis, minutes). (Fig. 1a, b, scale bar 250 μm)