Confocal microscopy analysis of popliteal LNs was performed as previously described28 . The following primary Abs were used for staining: rat anti-B220 (RA3-6B2), rabbit anti-GFP (Invitrogen). Images were acquired on an inverted Leica microscope (SP8, Leica Microsystems) with a motorized stage for tiled imaging using a HC PL APO CS2 20X objective (NA 0.75). To minimize fluorophore spectral spill over, we used the Leica sequential laser excitation and detection modality. B cell follicles were defined based on the positioning of polyclonal B cells or by B220 staining. For 3D imaging acquisition, 10 xy stacks (1024 x 1024 pixel) sampled with 2 μm z spacing were acquired to provide image volumes that were 20 μm in depth.
Hc pl apo cs2 20x objective
Manufactured by Leica
The HC PL APO CS2 20X objective is a high-performance objective lens designed for use in microscopy applications. It provides a magnification of 20X and is part of the Leica HC series. The objective is optimized for chromatic aberration correction and features a plan-apochromatic design.
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2 protocols using hc pl apo cs2 20x objective
1
Popliteal LN Immune Imaging by Confocal Microscopy
2
Popliteal LN Immune Imaging by Confocal Microscopy
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Confocal microscopy analysis of popliteal LNs was performed as previously described28 . The following primary Abs were used for staining: rat anti-B220 (RA3-6B2), rabbit anti-GFP (Invitrogen). Images were acquired on an inverted Leica microscope (SP8, Leica Microsystems) with a motorized stage for tiled imaging using a HC PL APO CS2 20X objective (NA 0.75). To minimize fluorophore spectral spill over, we used the Leica sequential laser excitation and detection modality. B cell follicles were defined based on the positioning of polyclonal B cells or by B220 staining. For 3D imaging acquisition, 10 xy stacks (1024 x 1024 pixel) sampled with 2 μm z spacing were acquired to provide image volumes that were 20 μm in depth.
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