Streptavidin af 488 conjugate

Tumors and mammary glands were fixed in 4% paraformaldehyde and paraffin embedded. When appropriate, control sections were treated with hyaluronidase (16 U/mL) for 30 min in a humidity chamber at 37 °C. Sections were then stained with either hematoxylin and eosin (H&E) or biotinylated hyaluronan binding-protein (HABP, MilliporeSigma #385,911 at 1:100) for 1 h at room temperature. Sections were incubated in Streptavidin AF 488 conjugate (at 1:500, FisherScientific #S11223) secondary antibodies for 1 h at room temperature in a humidity chamber: Tissues were mounted with a coverslip using ProLong Gold Antifade DAPI (Invitrogen, #P36931).

Tumors were fixed in 4% paraformaldehyde and paraffin embedded. When appropriate, control sections were treated with hyaluronidase (16 U/mL) for 30min, in a humidity chamber at 37 °C. Sections were then stained with either hematoxylin and eosin (H&E), F4/80 (#MCA497GA at 1:100, Biorad, Hercules, CA, USA) and CD206 (Abcam #ab64693 at 1:1000, Cambridge, UK) overnight at 4 °C, or biotinylated HABP (MilliporeSigma #385911 at 1:100), for 1 h at room temperature. Sections were incubated in the appropriate secondary antibodies for 1 h at room temperature in a humidity chamber: Streptavidin AF 488 conjugate (at 1:500, FisherScientific #S11223, Waltham, MA, USA), AF 594 (at 1:400, Invitrogen #A11007), and AF 488 (at 1:400, Fisher Scientific #A21206). Tissues were coverslipped with ProLong Gold Antifade DAPI (Invitrogen, #P36931).