Dual-Labeling Immunohistochemistry in Mouse Brain

After being deeply anesthetized with Ketamine and Xylazine, mice were perfused with phosphate buffer saline (PBS) and 4% paraformaldehyde in PBS. Brains were post-fixed overnight with 4%A PFA/PBS, embedded in 2% agarose /PBS, and then sectioned at 50 μm with a vibratome (Leica, Buffalo Grove, IL VT1000S). The following antibodies were used for immunohistochemistry: anti-GFP rabbit (Life Technologies, Thermo Fisher, Waltham, MA A-11122), anti-GFP chicken (Aves labs, Tigard, OR GFP-1020), anti-HA rat (Roche diagnostics, Indianapolis, IN clone 3F10). Whole slide images were taken with a microscope with 5x objective and XY-stage controlled by μManger (https://micro-manager.org). Grid/Collection stitching Fiji plugin (Preibisch et al., 2009 (link)) was used for image assembly. We followed the dual-color in situ protocol described in BraInSitu web site (http://www.nibb.ac.jp/brish/indexE.html) (Watakabe et al., 2006 (link)).

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Shima Y., Sugino K., Hempel C.M., Shima M., Taneja P., Bullis J.B., Mehta S., Lois C, & Nelson S.B. (2016). A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types. eLife, 5, e13503.

Publication 2016

VT1000S anti-GFP rabbit anti-GFP chicken anti-HA rat

Agarose Antibodies Aves Brains Buffalo Buffer Chicken Clone Diagnostics Immunohistochemistry Ketamine Mice Microscope Paraformaldehyde Phosphate Rabbit Saline Xylazine

Corresponding Organization : Brandeis University

Other organizations : Janelia Research Campus, Howard Hughes Medical Institute, Galenea (United States), California Institute of Technology

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Anesthesia with Ketamine and Xylazine

dependent variables

Localization and expression of GFP and HA proteins

control variables

Phosphate buffer saline (PBS)
4% paraformaldehyde in PBS
2% agarose in PBS
Vibratome sectioning at 50 μm
Antibodies used for immunohistochemistry: anti-GFP rabbit, anti-GFP chicken, anti-HA rat
Whole slide imaging at 5x objective
Image assembly using Grid/Collection stitching Fiji plugin
Dual-color in situ protocol from BraInSitu website

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