Mice were euthanized by carbon dioxide asphyxiation. Tumors were dissected and fixed with 10% formalin overnight, transferred to 70% ethanol, and then embedded in paraffin. 3μM thick sections were cut and stained with H&E. Immunohistochemistry on paraffin-embeded sections was performed using antibodies against Nqo1 (1:100, Sigma, HPA007308), phospho-histone-H3 (1:100, Ser10; Cell Signaling, #9701), 8-oxo-deoxyguanosine (1:100, Abcam, #ab48508), and cleaved caspase3 (1:200, Cell Signalin, #9579). Chromogenic IHC was performed on a Leica Bond RX and stained slides were imaged on a Leica SCN400 F whole-slide scanner. For Nqo1, 8-oxo-deoxyguanosine, and phospho-histone-H3 staining antigen retrieval was performed using antigen retrieval buffer pH6 (Leica) for 20 min and for cleaved caspase3 staining, antigen retrieval buffer pH9 (Leica) was used for 20min. For detection, Leica Bond Polymer Refine Detection secondary antibody (Leica, #DS9800) was used according to manufacturer’s protocol for Nqo1, phosphor-histone-H3 and cleaved caspase3. 8-oxo-deoxyguanosine was detected with the Biocare MM HRP-polymer secondary antibody (Biocare, #MM620H) according to manufacturer’s protocol.
Antigen retrieval buffer ph6
Manufactured by Leica
Antigen retrieval buffer pH6 is a solution used in immunohistochemical and immunocytochemical techniques to unmask or expose antigenic sites in fixed tissue or cell samples. It is designed to have a pH of 6.0, which is suitable for certain types of antigen retrieval.
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3 protocols using antigen retrieval buffer ph6
1
Immunohistochemical Characterization of Tumor Samples
2
Lung Tumor Histological Characterization
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Mice were euthanized by carbon dioxide asphyxiation. Lung with tumors were dissected, infused, and fixed with 10% formalin overnight; transferred to 70% ethanol; and then embedded in paraffin. Sections (3 mM thick) were cut and stained with H&E. Immunohistochemistry on paraffin-embedded sections was performed using antibodies against Nqo1 (1:100; Sigma-Aldrich, HPA007308) and G6pd (1:5000; Abcam, ab993). Chromogenic IHC was performed on a Leica Bond RX, and stained slides were imaged on a Leica SCN400 F whole-slide scanner. For Nqo1 and G6pd staining, antigen retrieval was performed using antigen retrieval buffer pH 6 (Leica) for 20 min. For detection, Leica Bond Polymer Refine Detection secondary antibody (Leica, no. DS9800) was used according to the manufacturer’s protocol for Nqo1 and G6pd.
3
Quantitative analysis of NQO1 in lung tumors
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Mice were euthanized by lethal doses of ketamine and xylazine. Lungs were inflated and incubated overnight at room temperature (RT) with 10% formalin. They were subsequently transferred to 70% ethanol, and subsequently embedded in paraffin. 5uM sections were stained with H&E or subjected to other immunohistochemical staining. For immunohistochemistry (IHC) we used antibody against NQO1 (1:100, HPA007308, Sigma-Aldrich). Immunohistochemistry was performed on a Leica Bond RX and slides were imaged on a Leica SCN400F whole slide scanner. For NQO1, antigen retrieval was performed using antigen retrieval buffer pH=6 (Leica) for 20min. For detection, Leica Bond Polymer Refine Detection secondary antibody was used according to manufacturer’s protocol. Total tumor lung area was quantified via H&E-stained slides using QuPath software.103 (link) Tumor burden and IHC analyses were done in a blinded fashion.
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