Immunofluorescence Staining of 3D Cell Cultures

MCF10A-5E and MCF10DCIS.com 3D cultures were embedded at day 10 of morphogenesis, and 5-μm sections were cut and mounted on Superfrost Plus slides (Fisher). For clinical samples, paraffin tissue sections were dewaxed and antigens retrieved on a PT Link (Dako) with low-pH EnVision FLEX Target Retrieval Solution (Dako) for 20 min at 97°C. Immunofluorescence on cryosections and antigen-retrieved slides was performed as previously described (20 (link)) with the following primary antibodies: NRF2 (Santa Cruz #sc-13032, 1:100), phospho-Rb (Cell Signaling #8516, 1:1600), HIF-1α (Cell Signaling #79233, 1:800), p53 (Santa Cruz #sc-126, 1:200). Slides were incubated the next day for 1 hour in the following secondary antibodies: Alexa Fluor 555-conjugated goat anti-rabbit (1:200; Invitrogen), Alexa Fluor 647-conjugated goat anti-mouse (1:200; Invitrogen).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Pereira E.J., Burns J.S., Lee C.Y., Marohl T., Calderon D., Wang L., Atkins K.A., Wang C.C, & Janes K.A. (2020). Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies. Science signaling, 13(627), eaba4200.

Publication 2020

Superfrost Plus slides PT Link EnVision FLEX Target Retrieval Solution phospho-Rb HIF-1α Alexa Fluor 555-conjugated goat anti-rabbit Alexa Fluor 647-conjugated goat anti-mouse

Alexa fluor 555 Alexa fluor 647 Antibodies Antigen Cryosections Goat Immunofluorescence Morphogenesis Mouse Nrf2 Paraffin Rabbit Tissue

Corresponding Organization : University of Virginia

Other organizations : California State University, Channel Islands, National Tsing Hua University

Top 5 similar protocols

Variable analysis

independent variables

Embedding time of MCF10A-5E and MCF10DCIS.com 3D cultures (at day 10 of morphogenesis)

dependent variables

Expression of NRF2, phospho-Rb, HIF-1α, and p53 proteins

control variables

Thickness of tissue sections (5-μm)
Type of slides used (Superfrost Plus slides, Fisher)
Antigen retrieval method (low-pH EnVision FLEX Target Retrieval Solution, Dako, for 20 min at 97°C)
Primary antibody concentrations (NRF2 1:100, phospho-Rb 1:1600, HIF-1α 1:800, p53 1:200)
Secondary antibody concentrations (Alexa Fluor 555-conjugated goat anti-rabbit 1:200, Alexa Fluor 647-conjugated goat anti-mouse 1:200)

controls

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

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!