3D FISH Analysis of Igκ Locus

Using our published techniques (15 (link), 16 (link), 30 (link)), pre-B cells were processed for 3D DNA FISH. Briefly, probes for 3D FISH were prepared from bacterial artificial chromosomes (BACs). We used RP23-101G13, RP23-26A6, and RP24-387E13, which correspond to the 5′, middle, and the 3′ region of the Igκ locus, respectively. To make probes for each slide, 1 μg BAC DNA samples were labeled by nick translation with ChromaTide Alexa Fluor 488–5-deoxyuridine triphosphate (dUTP), ChromaTide Alexa Fluor 594–5-dUTP (Invitrogen), or Cy5-dUTP (GE Healthcare). Hybridization conditions were as described previously (15 (link), 16 (link), 30 (link)). FISH signals were analyzed by Leica TCS SP5 confocal microscopy with Z slice-sections separated by 0.3 μm, and the center-to-center distances between different hybridizing signals were measured using a plug-in of ImageJ software.

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.

Xiang Y., Park S.K, & Garrard W.T. (2014). A major deletion in the Vκ-Jκ intervening region results in hyper-elevated transcription of proximal Vκ genes and a severely restricted repertoire. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3746-3754.

Publication 2014

ChromaTide Alexa Fluor 594–5-dUTP Cy5-dUTP TCS SP5 confocal microscopy

Alexa 594 Alexa fluor 488 Bacterial artificial chromosomes Confocal microscopy Deoxyuridine triphosphate Fish Hybridization Pre b cells

Corresponding Organization : The University of Texas Southwestern Medical Center

Other organizations : Tianjin Medical University

Top 5 similar protocols

Variable analysis

independent variables

Probes for 3D FISH prepared from bacterial artificial chromosomes (BACs)
RP23-101G13, RP23-26A6, and RP24-387E13 BACs corresponding to the 5', middle, and 3' region of the Igκ locus, respectively

dependent variables

Center-to-center distances between different hybridizing signals

control variables

Hybridization conditions as described previously (15, 16, 30)
Preparation of probes by nick translation with ChromaTide Alexa Fluor 488–5-dUTP, ChromaTide Alexa Fluor 594–5-dUTP, or Cy5-dUTP

controls

No positive or negative controls were 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!