Alexa fluor 647 anti cd68

Mice were euthanized 7 days after surgery via CO₂ asphyxiation. Post-euthanasia, mouse vasculature was perfused with warm saline followed by 4% PFA until tissues were fixed. The entire spinotrapezius muscle was explanted and permeabilized overnight with 0.2% saponin, then blocked overnight in 10% mouse serum. For immunofluorescence, tissues were incubated at 4°C overnight in a solution containing 0.1% saponin, 5% mouse serum, 0.5% bovine serum albumin, conjugated fluorescent antibody Alexa Fluor 488 anti-desmin (1:200 dilution, Abcam), and Alexa Fluor 647 anti-CD68 (AbD Serotec) for macrophage visualization. Following immunostaining, tissues were washed 4 times for 30 minutes each in 0.2% saponin for the first two washes, 0.1% saponin for the third wash, and PBS for the final wash, and then mounted in 50/50 glycerol/PBS.

Mice were euthanized 3 or 7 days after surgery via CO₂ asphyxiation. For lectin-based labeling of the vasculature in CX3CR1^GFP/+ mice, Alexa Fluor 568 isolectin IB4 (1mg/mL, Life Technologies) was diluted (50μL of isolectin, 150μL saline) and administered by jugular vein injection 15 minutes prior to euthanasia. Post-euthanasia, mouse vasculature was perfused with warm saline followed by 4% PFA until tissues were fixed. The entire spinotrapezius muscle was explanted and permeabilized overnight with 0.2% saponin, then blocked overnight in 10% mouse serum. For immunofluorescence, tissues were incubated at 4°C overnight in a solution containing 0.1% saponin, 5% mouse serum, 0.5% bovine serum albumin, and the following conjugated fluorescent antibodies: PE anti-CD31 antibody (1:100 dilution, eBioscience), Alexa Fluor 488 anti-CD206 (1:200 dilution, ABD Serotec), and Alexa Fluor 647 anti-CD68 (1:200 dilution, ABD Serotec). For desmin immunostaining, identical tissue processing procedures were followed and muscles were incubated overnight with Alexa Fluor 488 anti-desmin (1:200 dilution, Abcam) alone or in combination with PE anti-CD31 and Alexa Fluor 647 anti-CD206 (1:200 dilution, BioLegend). Following immunostaining, tissues were washed 4 times for 30 minutes each, and then mounted in 50/50 glycerol/PBS.