RPPA technology was used to quantify NQO1 protein level in cells as previously described39 . Briefly, equal amounts of protein lysates were printed onto nitrocellulose covered slides. Four serial dilutions and two technical replicates per dilution were deposited for each sample. Arrays were revealed with an anti-NQO1 antibody (HPA007308 from Sigma). Quantification and normalization of RPPA data was performed using the NormaCurve method39 .
Hpa007308
Manufactured by Merck Group
HPA007308 is a laboratory equipment product manufactured by Merck Group. It is a general-purpose device designed for various scientific applications. The core function of this product is to perform specialized tasks in a laboratory setting. Further details on the intended use or specific capabilities of this equipment are not available.
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Lab products found in correlation
4 protocols using hpa007308
1
Quantifying NQO1 Protein Levels
2
Tissue Microarray Analysis of NQO1 Expression
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Tissue microarrays (TMAs) from the FFPE specimens of patients were commercially developed (Outdo Biotech). Rabbit polyclonal antibody against NQO1 (1:200, HPA007308, Sigma) was used according to the manufacturer's protocol. Immune staining was performed simultaneously on all arrays to eliminate interassay variation. Levels of NQO1 staining were evaluated using an H‐score method, as previously described,33 , 34 and results were recorded as staining intensity (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive) multiplied by the percent of tumor‐positive area (0%‐100%). Two observers (YY and JZ) blinded to the clinical status of the donor independently assessed the H‐score of each tissue dot, and the scores of the 2 observers were averaged for analysis. Any controversial cases (defined as a difference in IHC scores more than 10% of the average score) were jointly re‐evaluated until a consensus was reached.
3
Immunohistochemical Analysis of NRF2 and NQO1
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Immunohistochemistry was performed on a Leica Bond-III Autostainer, using the Bond Polymer Refine Detection System Kit (Leica Biosystem), according to the manufacturer’s instructions. Epitope retrieval was performed in ER2 buffer (EDTA based buffer and surfactant, pH = 9) for 30 min at 95°C for NRF2 staining and in ER1 buffer (EDTA based buffer and surfactant, pH = 6) for 20 min for NQO1 staining. Primary NRF2 antibody (Santa-Cruz Biotechnology A-10, sc-365949) was applied at 1:100 dilution for 1 h at room temperature. Primary NQO1 antibody (SIGMA-ALDRICH, HPA007308) was applied at 1:150 dilution for 20 min at room temperature. Slides were counterstained in hematoxylin and mounted in Pertex mounting medium (CellPath). Staining was categorized using a standard pathological system, with the staining intensity (+ to +++) and the percentage of tumor stained cells (0 to 100%). Samples were considered NRF2-high if nuclear staining intensity was +++ in more than 50% of tumor cells, and intermediate (+++/<50% and ++/>50%), low (++/<50% and +/>50%), or negative (+/<50% and 0).
4
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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