Alexa fluor 488 conjugated isolectin b4

Alexa Fluor 488 conjugated isolectin-B4 was purchased from Vector Labs, Burlingame, CA, and PermaFluorTM aqueous mounting medium from Thermo Fisher Scientific, Waltham, MA. All other chemicals and reagents were purchased from Sigma-Aldrich Corp., St. Louis, MO, and were of HPLC grade or the highest grade available.

Mice were anesthetized (150 mg/kg ketamine and 13 mg/kg xylazine) and treated by intravenous injection of 400 U heparin prior to thoracotomy. The right atrium was severed and the mice were maximally vasodilated by infusing, via the left ventricle, 20 ml normal saline containing 1 g/l adenosine, 4 mg/l papaverine and 100 μg/ml heparin followed by 10 ml 2% formalin. The skin was removed and entire hindlimbs were fixed in 10% formalin (RT, 24 hr). Calf and adductor muscles were dissected en block from fixed hindlimbs, dehydrated and embedded in paraffin. Cross sections (7 μm) were prepared and subjected to antigen retrieval using 1x antigen unmasking solution (Vector Labs). The sections were blocked and permeabilized in 10% normal goat serum, 0.1% Triton X-100 in PBS for (RT, 1 hr) and incubated with Alexa Fluor 488-conjugated IsolectinB4 (1:25, Vector Labs) and primary antibodies, mouse anti-smooth muscle actin (1:500, Sigma-Aldrich Cat# A5228, RRID:AB_262054) and rat anti-CD31 (1:50, BD Biosciences Cat# 558736, RRID:AB_397095) in 1% BSA in PBS (RT, 2 hr). Sections were washed 3 × 5 min each with PBS and incubated with Alexa Fluor 546-goat anti mouse and Alexa Fluor 488-goat anti rat antibodies in 1% BSA PBS for 1 hr at RT. Following washing as above, DNA was stained with DAPI, sections mounted in FluoromountG (Southern Biotech) and imaged on a TCS SP2 Leica confocal microscope.