Multiplex IHC staining was performed to examine the co-expression of ATAD2 and Ki-67 in OC tissue sections on a commercially obtained OC tissue microarray (OV241c; containing 12 primary OC tissues) from Alenabio using an Opal™ 4-Color Manual IHC kit (PerkinElmer, Inc.). Briefly, the sections were first incubated with a specific ATAD2 antibody (1:1,000; catalog no. HPA029424; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, followed by incubation with a goat anti-rabbit immunoglobulin G antibody (1:5,000; catalog no. NEL840001KT, PerkinElmer, Inc.) for 1 h at room temperature and subsequently labeled with Opal 520 fluorophore (PerkinElmer, Inc.). For Ki-67 staining, an anti-Ki67 antibody (1:1,000; catalog no. ZM-0166; OriGene Technologies, Inc.) was used at room temperature for 1 h and Opal 690 fluorophore (PerkinElmer, Inc.) was used for labeling. Finally, nuclei were stained with DAPI (PerkinElmer, Inc.) for 30 min at room temperature and mounted with anti-fade medium. The inForm 2.4.2 software (PerkinElmer, Inc.) was used to analyze the multispectral images.
Zm 0166
Manufactured by OriGene
The ZM-0166 is a lab equipment product. It is a centrifuge designed for general laboratory use. The device is capable of centrifuging samples at high speeds to separate components based on density differences.
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Lab products found in correlation
Opal 4 color manual ihc kit, by PerkinElmer (1 mentions)
Inform 2, by PerkinElmer (1 mentions)
Hpa029424, by Merck Group (1 mentions)
Opal 520 fluorophore, by PerkinElmer (1 mentions)
Opal 690 fluorophore, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by PerkinElmer (1 mentions)
Ab19071, by Abcam (1 mentions)
Haematoxylin, by Abcam (1 mentions)
2 protocols using zm 0166
1
Multiplex IHC for ATAD2 and Ki-67 in OC
2
Immunohistochemical Analysis of PSMA Expression
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To assess the expression of PSMA within the vascular endothelium, we also stained for an endothelial cell marker, CD34, to identify the vasculature. haematoxylin and eosin-stained slides of the 150 tissue samples were reviewed. The tissue sections (4-μm-thick) were mounted on glass slides, deparaffinized and rehydrated. The slides were stained for CD34 (1:200; ZM-0046; Origene) and PSMA (diluted 1:100; ab19071; Abcam). The proliferative index was evaluated in parallel with Ki-67 (1:200; ZM-0166; Origene).
For antigen retrieval, the samples underwent treatment in a pressure cooker for 1 min at 125°C in EDTA buffer (pH 8.0) and were then cooked for another 2 min and 30 s at 100°C. The samples were allowed to cool to room temperature before incubation with 3% hydrogen peroxide for 15 min. Subsequently, the sections were incubated overnight at 4°C with primary antibody and then incubated sequentially with secondary antibody (Zhong Shan Jin Qiao) and diaminobenzidine for 20, 20, and 10 min, respectively. Finally, the slides were counterstained with haematoxylin (Abcam), dehydrated and mounted. The appropriate positive controls were prepared.
For antigen retrieval, the samples underwent treatment in a pressure cooker for 1 min at 125°C in EDTA buffer (pH 8.0) and were then cooked for another 2 min and 30 s at 100°C. The samples were allowed to cool to room temperature before incubation with 3% hydrogen peroxide for 15 min. Subsequently, the sections were incubated overnight at 4°C with primary antibody and then incubated sequentially with secondary antibody (Zhong Shan Jin Qiao) and diaminobenzidine for 20, 20, and 10 min, respectively. Finally, the slides were counterstained with haematoxylin (Abcam), dehydrated and mounted. The appropriate positive controls were prepared.
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