Immunohistochemistry Protocol for CXCL13

Mouse pancreata were fixed and processed for histology and immunohistochemistry (IHC) as described previously(3 (link)). The IHC protocol was modified to detect mouse and human CXCL13, where blocking was done in 1× bovine free blocking solution (Vector) supplemented with 0.5% Tween-20, and 10% serum for 1 hour at room temperature, followed by incubation with the primary antibody diluted in 1× bovine free blocking solution overnight at 4°C. Secondary biotinylated rabbit-anti-goat antibody (Vector) was diluted in 1× bovine free blocking solution as well. The following primary antibodies were used: rabbit anti-GFP (#2956S, Cell Signaling), rat anti-B220 (#BDB557390, Fisher), rabbit-anti-vimentin (#5741P, Cell Signaling), mouse-anti-CD20 (#555677, BD Pharmingen), rabbit-anti-phospho Histone H3 (#06-570, Millipore), goat-anti-mouse CXCL13 and goat-anti-human CXCL13 (#AF470 and # AF801, both from R&D systems). At least 9 mice per experimental condition were analyzed for GFP staining and 6 mice per condition were analyzed for pHH3 staining. Slides were examined on a Nikon Eclipse 80i microscope.

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Pylayeva-Gupta Y., Das S., Handler J.S., Hajdu C.H., Coffre M., Koralov S.B, & Bar-Sagi D. (2015). IL-35 producing B cells promote the development of pancreatic neoplasia. Cancer discovery, 6(3), 247-255.

Publication 2015

Secondary biotinylated rabbit-anti-goat antibody rabbit anti-GFP rabbit-anti-vimentin mouse-anti-CD20 rabbit-anti-phospho Histone H3 goat-anti-mouse CXCL13 goat-anti-human CXCL13 Eclipse 80i microscope

Anti antibody Antibodies Antibody Bovine Cxcl13 Goat Histone h3 Human Immunohistochemistry Mice Microscope Pancreata Rabbit Serum Tween 20 Vector Vimentin

Corresponding Organization : New York University

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Protocol cited in 2 other protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

GFP staining
Phospho Histone H3 (pHH3) staining

control variables

Mouse and human CXCL13 detection
Blocking with 1× bovine free blocking solution supplemented with 0.5% Tween-20 and 10% serum
Incubation with primary antibodies diluted in 1× bovine free blocking solution overnight at 4°C
Use of secondary biotinylated rabbit-anti-goat antibody diluted in 1× bovine free blocking solution
Primary antibodies used: rabbit anti-GFP, rat anti-B220, rabbit-anti-vimentin, mouse-anti-CD20, rabbit-anti-phospho Histone H3, goat-anti-mouse CXCL13 and goat-anti-human CXCL13

controls

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

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