Confocal Microscopy Analysis of Popliteal Lymph Nodes

Confocal microscopy analysis of popliteal LNs was performed as previously described (37 (link)). The following primary Abs were used for staining: rat anti-B220 (RA3-6B2), rat anti-CD169 (AbD Serotec), rabbit anti-GFP (Invitrogen), rat anti-LCMV Nucleoprotein (VL-4, Bioxcell), rat anti-TCRβ (H57-597, BioLegend). The following secondary Abs were used for staining: Alexa Fluor 488-, 514-, 568- or 647-conjugated goat anti-rabbit or anti-rat IgG (Life Technologies), Alexa Fluor 647-conjugated chicken anti-rat IgG (Life Technologies). Images were acquired on an inverted Leica microscope (SP8, Leica Microsystems) with a motorized stage for tiled imaging. To minimize fluorophore spectral spillover, we used the Leica sequential laser excitation and detection modality. B cell follicles, interfollicular areas (IFA) and T cell areas were defined based on the positioning of naïve B cells and, in certain experiments, by B220 and TCRβ stainings, as described (44 (link)).

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Sammicheli S., Kuka M., Di Lucia P., de Oya N.J., De Giovanni M., Fioravanti J., Cristofani C., Maganuco C.G., Fallet B., Ganzer L., Sironi L., Mainetti M., Ostuni R., Larimore K., Greenberg P.D., de la Torre J.C., Guidotti L.G, & Iannacone M. (2016). Inflammatory monocytes hinder antiviral B cell responses. Science immunology, 1(4), eaah6789.

Publication 2016

rat anti-CD169 rabbit anti-GFP

Alexa fluor 488 Alexa fluor 647 Anti igg B cells Chicken Confocal microscopy Follicles Goat Lcmv Microscope Nucleoprotein Rabbit Stainings T cell Tcrβ

Corresponding Organization : Istituti di Ricovero e Cura a Carattere Scientifico

Other organizations : University of Basel, The San Raffaele Telethon Institute for Gene Therapy, Fred Hutch Cancer Center, Scripps Research Institute

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Variable analysis

independent variables

Rat anti-B220 (RA3-6B2)
Rat anti-CD169 (AbD Serotec)
Rabbit anti-GFP (Invitrogen)
Rat anti-LCMV Nucleoprotein (VL-4, Bioxcell)
Rat anti-TCRβ (H57-597, BioLegend)

dependent variables

B cell follicles
Interfollicular areas (IFA)
T cell areas

control variables

Leica sequential laser excitation and detection modality to minimize fluorophore spectral spillover

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

Annotations

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