Las af channel dye separation module

Paraffin-embedded sections (6–10 µm) were deparaffinized, and antigen retrieval was performed using a decloaking chamber (125°C, 30 s, Biocare Medical) with Borg Decloaker buffer (Biocare Medical). Tissues were blocked (0.1 M Tris, 0.3% Triton X-100, 1% BSA) for 1 hr at room temperature (RT), stained with titrated amounts of nonconjugated antibodies (α-granzyme B, α-CD8) overnight at 4°C, washed with PBS (3 × 20 minutes), and stained with the appropriate secondary antibodies for 2 hr at RT. After a second blocking step with a 1:1 mixture of normal mouse serum (1 hr at RT), tissues were stained with titrated amounts of directly conjugated antibodies (α-CD20, α-perforin). JoJo staining was performed after a final wash step, and the slides were mounted with Fluoromount G (SouthernBiotech). Images were collected on a Leica SP8 confocal microscope using a 20× 0.75 numerical aperture (NA) or 40× 1.30 NA objective with a 1.5× optical zoom at a density of 1,024 × 1,024 pixels. Additional images were captured using a 63× 1.40 NA objective. Fluorophore spillover was corrected by imaging single-stained tissues and creating a compensation matrix via the Leica LAS-AF Channel Dye Separation module (Leica Microsystems). Compensated images were analyzed with Imaris software (Bitplane Scientific).