After harvest, spleens were passed through a sucrose gradient (10% for 1
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).
h, 20% for 2 h, 30% for 3 h) and flash frozen in Tissue Freezing Medium (General
Data TFM-5) using a Gentle Jane Snap Freezer. 10 μm sections were cut and
dried for 45 min in a 37 °C dry incubator. The sections were soaked in
ice-cold Zinc Formalin Fixative (Sigma) for 15 min at −20°C then
washed in 1× PBS (3 min/ wash). Sections were blocked with 1% BSA in
1× PBS for 2 h at 25 °C. After a 5-min wash in 1× PBS,
sections were stained with the primary antibodies (in 1% BSA in 1× PBS)
for 24 h at 4 °C. Sections were washed in 1× PBS (3 min/ wash)
then mounted using hard set mounting medium for fluorescence (VECTASHIELD
H-1400). Sections were stained with B220-AF488 (5 μg/ml; clone
RA3–6B2, eBioscience), CD4-AF594 (10 μg/ml; clone GK1.5,
BioLegend). GL7-AF647 (6.67 μg/ml; BD Pharmingen), and CD138-BV421 (1.67
μg/ml; clone 281–2, BioLegend). Imaging was done using a Zeiss
LSM710 confocal microscope and processed using IMARIS x64 software (version
9.2.1).