Af647 donkey anti rabbit igg

In total, 0.8×10⁶ tumor cells were subcutaneously injected into C57BL/6 mice. After 14 days, tumor tissues were dissected, fixed in 4% paraformaldehyde for 24 h, and dehydrated in 30% sucrose for 48 h. For tissue section staining, tissues were embedded in Optimal Cutting Temperature (OCT) compound, frozen, and cryo-sectioned (15–20 microns). Sections were permeabilized for 30 min in PBS with 0.1% Triton X-100 (PBST) and blocked for 1 h in PBS with 3% BSA. To stain for CD8⁺ T cells, rabbit anti-mouse CD8a (Abcam, ab217344, clone: EPR21769, dilution: 1:500) was used as the primary antibody, and AF647-donkey anti-rabbit IgG (Jackson Immuno Research, 711-605-152, clone: 30-F11, dilution: 1:500) was used as the secondary antibody. Stained tissue sections were imaged on a Zeiss LSM800 confocal microscope. Microscopy data were analyzed using OlyVIA software (OLYMPUS).

The muscles were frozen in 2‐methylbutan directly after removal. All cross‐sections were prepared on a cryostat at a thickness of 10 μm and stored at −20°C. For fibre‐type staining, the sections were first rehydrated with phosphate‐buffered saline (PBS) and then blocked for 30 min with blocking solution [0.4% Triton X‐100, 10% goat serum (Biological industries, Beit Haemek, Israel) in PBS] followed by a washing step with PBS. The primary antibodies against MyHC 2b (1:100; #BF‐F3; DSHB, Iowa, USA), MyHC 2a (1:200; #SC‐71; DSHB), MyHC 1 (1:50; BA‐D5; DSHB), and laminin (1:160; #ab11575; Abcam) were diluted in PBS containing 10% goat serum (Biological industries) and added for 1 h at RT. After a washing step with PBS, the secondary antibodies were added containing AF488 Gt anti‐Ms IgM (1:100; Invitrogen, Carlsbad, CA, USA), AF568 Gt anti‐Ms IgG1 (1:100; Invitrogen), DL405 Gt anti‐Ms IgG2b (1:50; Jackson), and AF647 Donkey anti‐Rabbit IgG (1:200; Jackson) diluted in PBS containing 10% goat serum. The slides were again washed with PBS and then mounted with Vectashield Hardset (H‐1600; Vector Labs, Burlingame, CA, USA). The entire section was imaged on a Zeiss (Oberkochen, Germany) Axio Scan.Z1 Slide Scanner.