Formalin-fixed paraffin-embedded 10-µm sections were stained with anti-CD8 antibodies (1:300, clone RPA-T8) conjugated with phycoerythrin. Antigen was retrieved using a heated 10 mM Tris-ethylenediaminetetraacetate buffer (pH 9.0) before staining. For immunostaining, sections washed with PBS buffer (pH 7.4) were incubated in PBS containing 10% (vol/vol) FBS, 0.3 mM glycine, and 0.5% (vol/vol) Triton X-100 for 1 h, and stained with an antibody at the appropriate dilution overnight at room temperature. Finally, sections were washed with PBS containing 0.1% Tween and mounted in a medium containing Hoechst 33342. The images were acquired using an Eclipse Ti-E microscope with confocal module А1, CFI Plan Apo VC 20 × 0.75, and Apo TIRF Plan Fluor 63 × 1.49 (Nikon Corporation).
Apo tirf plan fluor 63 1
Manufactured by Nikon
The Apo TIRF Plan Fluor 63 × 1.49 is a high-numerical aperture objective lens designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It has a magnification of 63X and a numerical aperture of 1.49.
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Lab products found in correlation
2 protocols using apo tirf plan fluor 63 1
1
Immunostaining of CD8+ T cells
2
Visualizing CAR19 and PD1 Expression
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Cells were placed on poly-D-lysin coated glasses and fixed with 4% paraformaldehyde in PBS, washed with PBS, permeabilized in 0.1% Triton X-100/PBS/0.1% FBS for 30 min at 4°C, and blocked with PBS/1% FBS/0.1% Tween-20 (PFT). To identify CAR19 distribution cells were stained with CD19 CAR Detection Reagent (1:600 in PFT) for 12 h, then samples were washed with PFT and treated for 1 h with anti-biotin-PE (Thermo Fisher Scientific, 1:1000). To assess PD1 expression, samples were treated for 12 h with anti-PD1 antibodies, conjugated with FITC (1:500 in PFT). For nuclei counterstaining 10 µM Hoechst 33,342 was used. Images were captured on an Eclipse Ti-E microscope with the A1 confocal module (Nikon Corporation) and Apo TIRF Plan Fluor 63 × 1.49.
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