Antigen unmasking solution

LY6161 high-density lymphoma and normal lymph node tissue arrays were ordered from U.S. Biomax, Inc and stained using the following protocol. Tissue array slides were deparaffinized and hydrated through xylene and a graded alcohol series, rinsed for 5 minutes in water, and incubated for 5 minutes in 3% H₂O₂. Slides were then washed 2 times followed by antigen unmasking using 1x antigen unmasking solution (Vector Laboratories) for 30 minutes. Slides were allowed to cool, washed 3 times in 0.1% Tween-20 PBS buffer, and blocked for 30 minutes with 2.5% normal horse blocking serum (Vector Laboratories). Slides were then stained with antibodies directed against BCL10 (1:100), CD90 (1:10), CAV1 (1:100), and GILZ (1:50), overnight at 4°C. Slides were subsequently washed 3 times, incubated for 30 minutes with ImmPRESS^™ reagent (Vector Laboratories), and washed an additional 3 times. Staining was revealed in peroxidase substrate DAB solution, with rinsing in tap water. Slides were counterstained with Hematoxylin QS (Vector Laboratories) and mounted with permanent mounting medium (Sigma).

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.

Immunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories). Endogenous peroxidase activity was quenched with 3% H₂O₂. After blocked with 5% normal serum, the slides were incubated with primary antibodies in a humidified chamber overnight. The next day, the slides were incubated with appropriate secondary antibodies and ABC solution using ABC Elite kit (Vector Laboratories). The immunoreactivity was revealed by incubating the slides in DAB solution. Nuclear staining was performed using hematoxylin. The slides were dehydrated, cleared, and mounted. The images were acquired and analyzed by NIS Element software with Nikon microscope image system (Nikon Instruments).