Immunohistochemical staining (IHC) on tumor sections was performed as described previously (32 (link)–34 (link)), using antibodies against EPHA2 (Life Technologies; #347400), EGFRL858R (Cell Signaling Technology, #3197S), PCNA (BD Pharmingen, #555567), and vWF (DakoCytomation, #A0082). PCNA+ staining was quantified as the average percentage of PCNA+ nuclei relative to total nuclei (proliferation index). Apoptosis assays were performed using the Apoptag Red In Situ Apoptosis Detection Kit per the manufacturer’s protocol (Millipore). TUNEL+ staining was quantified as the percentage of TUNEL+ nuclei relative to total nuclei (apoptotic index). Tumor vessels were quantified by assessing the vWF+ vessels (pixels). Four fields of at least 5 independent tumors per genotype or treatment condition were assessed for all staining quantification. Biotin goat anti-rabbit (BD Pharmingen), anti-rabbit Cy3 (Jackson ImmunoResearch), retrievagen A (pH 6.0) (BD Pharmingen, #550524), streptavidin peroxidase reagents (BD Pharmingen, #51-75477E), and the liquid 3,3′-diaminobenzidine tetrahydrochloride substrate kit (Zymed Laboratories) were used for IHC. Cytoseal XYL (Richard Allan Scientific) or ProLong Gold antifade reagent with DAPI (Life Technologies) were used to mount slides.
Retrievagen a ph 6.0
Manufactured by BD
Retrievagen A (pH 6.0) is a laboratory reagent produced by BD. It is designed to maintain a pH of 6.0 during sample processing and preparation.
Automatically generated - may contain errors
Lab products found in correlation
Epha2, by Thermo Fisher Scientific (1 mentions)
Biotin goat anti rabbit, by BD (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Proliferating cell nuclear antigen (pcna), by BD (1 mentions)
Egfrl858r, by Cell Signaling Technology (1 mentions)
2 protocols using retrievagen a ph 6.0
1
Quantitative Analysis of Tumor Biomarkers
2
Immunohistochemical Staining of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Small pieces (~1cm x 1cm) of freshly harvested liver were immersion fixed in 10% neutral-buffered formalin for 24–48 hours and processed to paraffin for sectioning at 5 microns for routine H & E staining or IHC as previously described [165 (link)]. Primary and secondary antibodies and reagents and conditions used for IHC are listed in S6 Table . Antigen retrieval (Retrievagen A pH 6.0, BD Pharmingen) was performed in a microwave at 95°C for 10 minutes for phospho-S6, total S6 and pan-CK, 20 minutes for p53 and 50 minutes for c-Myc IHC. All primary antibody incubations were performed overnight at 4°C except for c-Myc which was performed for 48 hours at 4°C. All secondary antibody incubations were performed at room temperature for 1–2 hours and slides were incubated with streptavidin horseradish peroxidase (HRP) conjugate at room temperature for 20–60 minutes. 3-Amino-9-Ethylcarbazole (AEC) chromagen was prepared as recommended by the manufacturer. After color development, sections were either immediately cover-slipped (no counterstain) or counterstained with Hematoxylin QS.
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