Multicolor Immunofluorescence Analysis of Murine Spleen

Seven micrometer spleen and heart cryostat sections were air-dried and fixed in acetone. Primary mAbs specific for the following mouse epitopes were used for immunohistochemical/fluorescent staining: C4d (clone 16-D2 Abcam, Cambridge, UK), NK1.1 (PK136, Abcam) CD68 (ER-HR3, Abcam), mucosal addressin cell adhesion molecule (MAdCAM-1; clone MECA-367, Abcam), CD31 (Novus Biologicals, CO, USA), α-smooth muscle Actin (Thermo Fisher Scientific), and IgG-FITC (BD Biosciences, San Diego, CA, USA). Splenic GCs were identified by double-labeling sections with rat anti-mouse B220-APC (clone RA3-6B2) and rat anti mouse GL7-FITC (both BD Biosciences). Numbers of GL7⁺ GCs were expressed as a percentage of total B220⁺ lymphoid follicles (44 (link)). CD4 T cells within GCs were located with rat anti-mouse CD4-biotin (BD Biosciences) & Streptavidin-Alexa Fluor 555 (Thermo Fisher Scientific). Confocal images were captured with a Leica SP5 confocal microscope using LAS AF software, version 2.7.2.9586 (Leica Microsystems, Wetzlar, Germany).

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Chhabra M., Alsughayyir J., Qureshi M.S., Mallik M., Ali J.M., Gamper I., Moseley E.L., Peacock S., Kosmoliaptsis V., Goddard M.J., Linterman M.A., Motallebzadeh R, & Pettigrew G.J. (2019). Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury. Frontiers in Immunology, 9, 3038.

Publication 2018

C4d (clone 16-D2 α-smooth muscle Actin IgG-FITC rat anti-mouse B220-APC (clone RA3-6B2) rat anti mouse GL7-FITC rat anti-mouse CD4-biotin Streptavidin-Alexa Fluor 555 Leica SP5 confocal microscope LAS AF software,

Acetone Actin Alexa fluor 555 Biologicals Biotin Cd4 t cells Cell adhesion molecule Clone Confocal microscope Epitopes Fitc Follicles Heart Lymphoid Mabs Madcam 1 Meca Mouse Mucosal Novus Smooth muscle Splenic Streptavidin

Corresponding Organization : University College London

Other organizations : Papworth Hospital, Cambridge University Hospitals NHS Foundation Trust, Babraham Institute

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Variable analysis

independent variables

Primary mAbs specific for the following mouse epitopes: C4d, NK1.1, CD68, MAdCAM-1, CD31, α-smooth muscle Actin, IgG-FITC

dependent variables

Numbers of GL7+ GCs expressed as a percentage of total B220+ lymphoid follicles
CD4 T cells within GCs

control variables

Seven micrometer spleen and heart cryostat sections
Air-dried and fixed in acetone
Rat anti-mouse B220-APC (clone RA3-6B2) and rat anti mouse GL7-FITC
Rat anti-mouse CD4-biotin and Streptavidin-Alexa Fluor 555

controls

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

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