Alexa fluor 647 anti mouse cd68

Ligation or sham surgery was performed as described above. Three days post-surgery, mice were humanely euthanized and samples of adipose tissue from the proximal and distal regions of each inguinal fat pad were harvested and fixed overnight in 4% PFA at 4°C. Samples were washed three times with PBS and submerged in 100 µL of 0.3% (v/v) Triton X-100/PBS for 3 hours at room temperature to permeabilize the tissue. Following permeabilization, the tissue was submerged in either (1) 100 µL of Alexa Fluor 488 conjugated isolectin (1:300) and Alexa Fluor 647 anti-mouse CD68 (AbD Serotec, Raleigh, NC, Clone FA-11) (1:200) diluted in 0.3% Triton X-100/PBS, or (2) 100 µL of Alexa Fluor 546 conjugated isolectin (1:300), Alexa Fluor 647 anti-mouse CD68 (AbD Serotec, Raleigh, NC, Clone FA-11) (1:200), and Alexa Fluor 488 anti-mouse CD206 (AbD Serotec, Raleigh, NC, Clone MR5D3) (1:200) diluted in 0.3% Triton X-100/PBS. Samples were incubated in antibody solution on a rocker at 4°C overnight protected from light. Following staining, samples were washed five times with 0.3% Triton X-100/PBS for five minutes per wash. Samples were mounted on glass slides. 200× images (at least four unique FOVs for each sample) were acquired using confocal microscopy, and CD68⁺ and CD206⁺ cells were quantified using ImageJ.