Visualizing Lymphoid Tissue Microarchitecture

Tissue fixation and cryosectioning was performed as described (8 (link)). Sections were rehydrated in PBS for 20 mins, blocked with 5% FBS in PBS/0.1% TWEEN®20 for 20 mins, and stained with primary antibodies for 1 h. Sections were washed 5 times with PBS/0.1% TWEEN®20 and secondary antibodies applied for 1 h. Slides were mounted with Fluorescence Mounting Medium (DAKO). Images were acquired on a Zeiss Z1 Observer fluorescent microscope equipped with Colibri LED light sources, or with Leica SP8 confocal microscope. The following primary antibodies were used: anti-MAdCAM1 (MECA-367, Biolegend), rabbit anti-RFP (polyclonal, Rockland), CD3-biotin (1452C11), CD35-biotin (8C12, BD). The following secondary reagents were used: donkey anti-rat-IgG (H+L) Alexa Fluor 488 (Jackson Immuno Research), donkey anti-rabbit-IgG (H+L) Alexa Fluor 555 (Thermo Fisher Scientific), Streptavidin-Alexa Fluor 488 and Streptavidin-Alexa Fluor 555 (Thermo Fisher Scientific). Conjugated antibodies: IgD-Alexa Fluor 647 (11-26c.2a, Biolegend), CD21/35-FITC (7E9). Nuclei were labeled with DAPI. Images were analyzed with ImageJ (69 (link)) and Zen (Zeiss).

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Zehentmeier S., Lim V.Y., Ma Y., Fossati J., Ito T., Jiang Y., Tumanov A.V., Lee H.J., Dillinger L., Kim J., Csomos K., Walter J.E., Choi J, & Pereira J.P. (2022). Dysregulated stem cell niches and altered lymphocyte recirculation cause B and T cell lymphopenia in WHIM syndrome. Science immunology, 7(75), eabo3170.

Publication 2022

Fluorescence Mounting Medium SP8 confocal microscope (MECA-367, donkey anti-rabbit-IgG (H+L) Alexa Fluor 555 Streptavidin-Alexa Fluor 488 Streptavidin-Alexa Fluor 555 Zen

Alexa fluor 488 Alexa fluor 555 Alexa fluor 647 Anti igg Antibodies Biotin Cd35 Confocal microscope Dapi Donkey Fitc Fluorescence Light Meca Microscope Nuclei Rabbit Streptavidin Tissue fixation Tween 20

Corresponding Organization : Yale University

Other organizations : The University of Texas Health Science Center at San Antonio, Korea University, University of South Florida, Johns Hopkins University, Johns Hopkins All Children's Hospital

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Variable analysis

independent variables

Tissue fixation and cryosectioning protocols
Primary antibodies used: anti-MAdCAM1, rabbit anti-RFP, CD3-biotin, CD35-biotin
Secondary reagents used: donkey anti-rat-IgG Alexa Fluor 488, donkey anti-rabbit-IgG Alexa Fluor 555, Streptavidin-Alexa Fluor 488, Streptavidin-Alexa Fluor 555, IgD-Alexa Fluor 647, CD21/35-FITC

dependent variables

Fluorescence microscopy images acquired using Zeiss Z1 Observer or Leica SP8 confocal microscope

control variables

Rehydration in PBS for 20 mins
Blocking with 5% FBS in PBS/0.1% TWEEN®20 for 20 mins
Washing 5 times with PBS/0.1% TWEEN®20
Mounting with Fluorescence Mounting Medium (DAKO)

controls

Positive control: None specified
Negative control: None specified

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