Ab106732

Eyes enucleated from OIR mice were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and cut at 3-μm thickness. After deparaffinization, rehydration, antigen retrieval by citric acid, and blocking with 5% skim milk, the sections were incubated with the primary antibodies overnight at 4°C, and the secondary antibodies were added for 1 hr at room temperature. Nuclei were counterstained with Hoechst 33342 (Molecular Probes). After washing with PBS, the slides were coverslipped with an aqueous mounting medium (Thermo). A fluorescent microscope (BZ-9000; Keyence) was used to examine and analyze the slides.
The primary antibodies were POSTN (MAB 3548: 5 μg/mL; R&D Systems), CD31 (550274: 1:50 dilution; BD Biosciences), αSMA (F3777: 1:250 dilution; Sigma-Aldrich), F4/80 (MCA497: 1:100 dilution; AbD Serotec), CD206 (1:100 dilution; Biolegend), IL-13 (ab106732: 1:100 dilution; Abcam), and CD4 (sc-1140: 1:100 dilution; Santa Cruz). The secondary antibodies were Alexa Fluor 488 and 647 (1:100 dilution; Molecular Probes).

Paraffin sections of human skin were deparaffinized, rehydrated, and permeabilized in phosphate‐buffered saline with 0.2% Triton X‐100 (PBS‐T), and pre‐incubated in PBS containing 5% normal donkey serum (blocking solution) at room temperature for 1 h. Specimens were then incubated with antibody to IL‐20 (Invitrogen, AB_2720694, PA5‐76967, 1:100), IL‐20R1 (Abcam, RRID:AB_2750843, ab203196, 1:100), IL‐20R2 (Abcam, RRID:AB_10678407, ab95824, 1:200), or PGP9.5 (Abcam, RRID:AB_1269733, ab72911, 1:500), or IL‐13 (Abcam, RRID:AB_10867235, ab106732, 1:500) in blocking solution (4°C, overnight). The specimens were washed with PBS and incubated with Alexa 488 or Alexa 594 conjugated IgG. Following removal of unbound secondary antibody, specimens were mounted using prolong anti‐fade reagents containing (4′,6‐diamidino‐2‐phenylindole) DAPI. Images were taken by IX73 Olympus inverted microscope using the CellSens Dimension Imaging software.