Immunohistochemistry and In Situ Hybridization Protocols

IHC was performed as previously described^{69 (link)} using a 1:400 antibody dilution for PKM2 (C-11), 1:600 dilution for PKM1 (Cat# NBP2-14833, Novus Biologicals; Littleton, CO), and 1:250 dilution for GLUT1 (AbCam; Cat# ab652). ISH was performed as described^{70 (link)} using LNA^™ microRNA ISH miR-122 optimization kit (Cat# 90003, Exiqon; Woburn, MA) followed by incubation of sheep anti-digoxigenin-AP (Cat# 11093274910, Roche Diagnostics; Mannheim, Germany), and developed with NBT:BCIP (Cat# SK-5400, Vector Laboratories; Burlingame, CA) at 30°C overnight. Nuclear fast red was used to counterstain nuclei (Cat# H-3403, Vector Laboratories).

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Fong M.Y., Zhou W., Liu L., Alontaga A.Y., Chandra M., Ashby J., Chow A., O’Connor S.T., Li S., Chin A.R., Somlo G., Palomares M., Li Z., Tremblay J.R., Tsuyada A., Sun G., Reid M.A., Wu X., Swiderski P., Ren X., Shi Y., Kong M., Zhong W., Chen Y, & Wang S.E. (2015). Breast cancer-secreted miR-122 reprograms glucose metabolism in pre-metastatic niche to promote metastasis. Nature cell biology, 17(2), 183-194.

Publication 2015

NBP2-14833 LNA™ microRNA ISH miR-122 optimization kit sheep anti-digoxigenin-AP NBT:BCIP Nuclear fast red

Antibody Biologicals Diagnostics Digoxigenin Dilution Glut1 Microrna Novus Nuclei Sheep Vector

Corresponding Organization :

Other organizations : City of Hope, University of California, Riverside, City Of Hope National Medical Center, Tianjin Medical University Cancer Institute and Hospital

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Variable analysis

independent variables

Antibody dilution for PKM2 (C-11)

dependent variables

Protein expression levels of PKM2 (C-11)
Protein expression levels of PKM1
Protein expression levels of GLUT1
Expression levels of miR-122

control variables

Experimental protocols for IHC and ISH as described in previous publications (references 69 and 70)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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