Anti cd31 alexa488

Lungs were embedded in Tissue-Tek^®,O.C.T. Compound (SAKURA Finetek, Tokyo) and frozen in liquid nitrogen for preparation of cryosections. Frozen lung tissues were cut into 6 μm thick sections, immunostained and visualized by confocal microscopy (Fluoview FV 10i, Olympus, Tokyo). The sections were fixed in acetone for 10 min, blocked with Block Ace (Dainippon Sumitomo Pharma, Tokyo) for 10 min, and incubated with the primary and secondary antibodies for 60–120 min. The following antibodies were used for immunostaining: anti-CD31-Alexa488 (BioLegend, San Diego, CA), anti-CD45-Alexa647 (BioLegend), anti-CD26 (R&D Systems, Minneapolis, MN), anti-α-SMA (Thermo Scientific, Waltham, MA), and anti-S100 calcium-binding protein A4 (S100A4) (Abcam, Cambridge, UK).

Flow cytometry was performed using a FACSCalibur apparatus (Becton Dickinson, Franklin Lakes, New Jersey, USA) and results were analyzed with the CellQuest Pro software (Becton Dickinson). HMVEC were trypsinized and stained in PBS with 1% BSA with directly coupled antibodies: anti-podoplanin Alexa 647 (Biolegend, San Diego, CA, USA) and anti-CD31 Alexa 488 (Biolegend) (Table 1). Corresponding isotypic antibodies were used as controls. Gating strategy was performed according to FSC and SSC parameters. 10 000 events were acquired.Table 1

Primary Antibodies used for immunolabeling.

Antibody	Host	Type	Company	Catalog number	Dilution
P-MEL	Rabbit	IgG	Thermo Fisher	PA5-32491	1/100
CD31	Sheep	IgG	R&D Systems	AF806	1/100
Podoplanin	Mouse	IgG1	Angio-Bio Co.	11-003	1/50
LYVE-1	Rabbit	IgG	Fitzgerald	70R-LR003	1/100
CD31 Alexa 488	Mouse	IgG1,κ	Biolegend	303109	1/200
Podoplanin Alexa 647	Rat	IgG2a,𝜆	Biolegend	337007	1/200
Ki67	Mouse	IgG1,κ	BD Pharmingen	556003	1/700
Cleaved Caspase 3	Rabbit	IgG	Millipore	PC679	1/100
Fibronectin	Mouse	IgG1	ATCC hybridoma	CRL-1606	1/100
Laminin 332	Mouse	IgG1	Millipore	MAB19562	1/100
Collagen type VII	Mouse	IgG	Millipore	MAB1345	1/200
Filaggrin	Mouse	IgG1,κ	Abcam	Ab3137	1/500
Involucrin	Mouse	IgG1	Sigma	I9018	1/1000