Dab staining

Manufactured by Thermo Fisher Scientific

DAB staining is a widely used immunohistochemical technique that employs the chromogen 3,3'-diaminobenzidine (DAB) to visualize specific target proteins in tissue samples. This process involves the enzymatic conversion of DAB, resulting in the deposition of a brown-colored precipitate at the site of the target antigen.

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Lab products found in correlation

Lsm 510, by Zeiss (2 mentions) Tgfbr2, by Abcam (1 mentions) Foxa1, by Santa Cruz Biotechnology (1 mentions) Alexa flour secondary antibody, by Thermo Fisher Scientific (1 mentions) Vectashield containing dapi, by Vector Laboratories (1 mentions) Asf1a, by Cell Signaling Technology (1 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions)

4 protocols using dab staining

Immunohistochemical Analysis of GABPA and TGFBR2

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Paraffin embedded slides were deparaffinized and rehydrated followed by antigen-retrieval using citric acid buffer. Endogenous peroxidase was deactivated by H₂O₂. Slides were blocked using 10% goat serum and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 45 min at room temperature, DAB staining (Thermo Fisher Scientific) was used to detect the antigen–antibody binding. The primary antibodies used were: GABPA (ProteinTech) and TGFBR2 (Abcam). The slides were examined by two of the co-authors (ZF and NZ) and mean values of GABPA and TGFBR2 positive cells were presented based on the results from two observers. For each slide, a total of 200 cells in two fields were analyzed.

Fang Z., Zhang N., Yuan X., Xing X., Li X., Qin X., Liu Z., Neo S., Liu C., Kong F., Björkholm M., Fan Y, & Xu D. (2022). GABPA-activated TGFBR2 transcription inhibits aggressiveness but is epigenetically erased by oncometabolites in renal cell carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 41, 173.

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Quantifying GABPA and FoxA1 Expression

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Paraffin-embedded slides were deparaffinized and rehydrated followed by antigen retrieval using citric acid buffer. Endogenous peroxidase was deactivated by H₂O₂. Slides were blocked using 10% goat serum and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 45 min at room temperature, DAB staining (Thermo Fisher Scientific) was used to detect the antigen–antibody binding. The primary antibodies used were: GABPA (Cell Signaling Technology) and FoxA1 (Santa Cruz Biotechnology). The slides were examined by two of the coauthors (YG and DX) and mean values of GABPA and FoxA1 positive cells were presented based on the results from two observers. For each slide, a total of 200 cells in two fields were analyzed.

Guo Y., Yuan X., Li K., Dai M., Zhang L., Wu Y., Sun C., Chen Y., Cheng G., Liu C., Strååt K., Kong F., Zhao S., Bjorkhölm M, & Xu D. (2019). GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer. Cell Death and Differentiation, 27(6), 1862-1877.

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Immunohistochemical Analysis of Murine Tissues

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Mice were perfused with 4% paraformaldehyde in PBS prior to tissue collection. Prostate, kidney, lung and liver tissues were post-fixed and embedded either in paraffin or frozen blocks. Tissues were sliced (10 μm) for immunohistochemistry, H&E and Ki67 staining (Abcam, Cambridge, MA) followed by DAB staining (Thermo, Grand Island, NY). For immunostaining, frozen sections were washed with PBS, followed by incubation with 1% triton X-100 for 15min for permeabilization. Sections were blocked in 5% BSA in TBS for 30min followed by incubation with primary antibodies (Abcam, 1: 100 dilutions) overnight. Slides were washed with chilled PBS and incubated for another hour with Alexa Flour secondary antibodies at room temperature (Thermo, Grand Island, NY). After washing slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). Slides were viewed under the confocal imaging microscope (LSM510, Carl Zeiss, Germany).

Gao F., Alwhaibi A., Sabbineni H., Verma A., Eldahshan W, & Somanath P.R. (2017). Suppression of Akt1- β-catenin pathway in advanced prostate cancer promotes TGFβ1-mediated epithelial to mesenchymal transition and metastasis. Cancer letters, 402, 177-189.

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Immunohistochemical Analysis of ASF1A

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Paraffin embedded slides were deparaffinized and rehydrated followed by antigen-retrieval using citric acid buffer. Endogenous peroxidase was deactivated by H₂O₂. Slides were blocked using 10% goat serum and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 45 mins at room temperature, DAB staining (Thermo Fisher Scientific) was used to detect the antigen-antibody binding. The primary antibodies used were: ASF1A (Cell Signaling Technology, Cat# 2990), PCNA (Santa Cruz Biotechnology, Cat# sc-7907), and E-cadherin (Cell Signaling Technology, Cat# 3195s). The slides were examined by two of the co-authors (XL and DX) and mean values of ASF1A positive cells were presented based on the results from two observers. For each slide, a total of 200 cells in two fields were analyzed.

Liang X., Yuan X., Yu J., Wu Y., Li K., Sun C., Li S., Shen L., Kong F., Jia J., Björkholm M, & Xu D. (2017). Histone Chaperone ASF1A Predicts Poor Outcomes for Patients With Gastrointestinal Cancer and Drives Cancer Progression by Stimulating Transcription of β-Catenin Target Genes. EBioMedicine, 21, 104-116.

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