Anti hamster biotin

M-ATOs were fixed in 4% Formaldehyde (Sigma-Aldrich, Cat#
F8775) for 30 minutes at room temperature followed by 3×10
min washes in PBST (0.3% Triton X-100) and a 1-hour block in PBST/BSA (2%
BSA). M-ATOs were stained with anti-CD8a (clone 53–6.7;
Biolegend), anti-mDLL4 (clone HMD4–1;
Biolegend), anti-CD4 (clone RM4–5;
Biolegend), and anti-GFP (clone FM264G; Biolegend) at a
1:100 dilution, and anti-CD3 (clone 145–2C11; Biolegend)
at a 1:50 dilution overnight at 4°C. Secondary antibodies
AlexaFluor-594-conjugated anti-rat IgG (H+L) (Jackson ImmunoResearch,
Cat# 712–585-150) or Alexa-Fluor-488-conjugated anti-rat
IgG (H+L) (Jackson ImmunoResearch, Cat# 712–485-153)
were added at a 1:200 dilution for 2 hours at room temperature. For
anti-mDLL4, anti-hamster biotin (Jackson ImmunoResearch, Cat#
127–065-160) was added at a 1:500 dilution for 2 hours at
room temperature, and then AlexaFluor-594-conjugated Streptavidin
(Jackson ImmunoResearch, Cat# 016–580-084) was added
at a 1:800 dilution for 30 minutes at room temperature. Each M-ATO was
mounted individually in Vectashield Antifade Mounting Medium (Vector
Laboratories, Cat# H1000) on a concavity microscope slide
(Fisher Scientific). Immunofluo-rescence images were
acquired on a Zeiss LSM 880 confocal microscope equipped with Airyscan and
Zen software (Zeiss).