Immunofluorescent Tissue Staining Protocol

Harvested mouse tissues were fixed in 2% paraformaldehyde for 2 h before being treated with 30% sucrose overnight for cryoprotection. The sucrose-treated tissue was embedded in OCT tissue-freezing medium and frozen in a 2-methyl butane liquid bath. Frozen blocks were processed, stained, imaged, and enumerated by QIM as described (Steinert et al., 2015 (link)). Tissues were stained with antibodies to the following markers: CD4 (GK1.5; BD Biosciences), CD90.1 (OX-7; BD Biosciences), CD45.1 (A20; BioLegend), B220 (RA3-6B2; BioLegend), CD8β (YTS156.7.7; BioLegend), and collagen-IV (goat anti-mouse polyclonal; Millipore). We counterstained with DAPI or SYTOX Green (Thermo Fisher Scientific) to detect nuclei. The following secondary antibodies were from Jackson ImmunoResearch: bovine anti-goat (polyclonal) and donkey anti-rat (polyclonal).

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Beura L.K., Fares-Frederickson N.J., Steinert E.M., Scott M.C., Thompson E.A., Fraser K.A., Schenkel J.M., Vezys V, & Masopust D. (2019). CD4+ resident memory T cells dominate immunosurveillance and orchestrate local recall responses. The Journal of Experimental Medicine, 216(5), 1214-1229.

Publication 2019

CD4 (GK1.5; CD90.1 (OX-7; CD45.1 (A20; B220 (RA3-6B2; CD8β (YTS156.7.7; collagen-IV DAPI SYTOX Green bovine anti-goat donkey anti-rat

Antibodies Bath Bovine Cd90 Collagen iv Dapi Donkey Frozen Goat Methyl butane Mouse Nuclei Paraformaldehyde Sucrose Sytox green Tissues

Corresponding Organization : University of Minnesota

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Variable analysis

independent variables

Fixation in 2% paraformaldehyde for 2 h
Cryoprotection in 30% sucrose overnight
Embedding in OCT tissue-freezing medium
Freezing in 2-methyl butane liquid bath

dependent variables

Staining with antibodies to CD4, CD90.1, CD45.1, B220, CD8β, and collagen-IV
Nuclei counterstaining with DAPI or SYTOX Green

control variables

Tissue processing, staining, imaging, and enumeration by QIM as described in Steinert et al., 2015

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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