Anti fsp 1

For analysis of thymic medulla and cortex by immunohistology, thymi from GCV treated TK⁻ and TK⁺ mice were fixed in 4% formalin and embedded in paraffin blocks. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by light microscopy. For immunofluorescence, serial sections (5 μm) from OCT-embedded frozen tissues or primary cultured cells were fixed in cold acetone or 4% polyoxymethylene and blocked in PBS/1% BSA, washed in PBS/0.05% Tween and incubated with optimal dilutions of fluorochrome-conjugated antibodies: Alexa Fluor® 488 anti-I-A/I-E, anti-CD31-PE (Biolegend), anti-CD11c-PE, anti-CD11b-FTIC (BD Pharmingen), and anti-F4/80-PE (eBioscience), or with first Abs: anti-cytokeratin 5, anti-FSP1, anti-ER-TR7 (Abcam), anti-cytokeratin 8 (Tromal-1; Developmental Studies Hybridoma Bank), Biotinylated UEA-1, anti-vimentin (BD Pharmingen), anti-α-SMA, anti-Pan-CK (Sigma, Cat no. C5992), anti-CD140a/PDGFRα (R&D Systems) and anti-MTS15 Ab for 2 h at room temperature before washing and incubating with secondary reagents: Alexa Fluor® 546 Goat anti-Rabbit/mouse IgG (H+L), Alexa Fluor® 488 Goat anti-Rat IgG (H+L) (Invitrogen), Dylight 488 Goat anti-Rabbit/mouse IgG (ZSGB-Bio) and streptavidin-PE (BD PharMingen). Control slides were incubated with isotype-matched Ig. Images were acquired with a two-photon microscopy (Carl Zeiss, Inc.).

Total protein was extracted and size-fractioned by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk in PBS with 0.1% Triton-X100, immunodetection was carried out using specific primary antibodies: anti-VCAM (Abcam), anti-ICAM (BioLegend), anti-p50 (Santa Cruz Biotechnology), anti-p84 (Santa Cruz Biotechnology), anti-p65 (Santa Cruz Biotechnology, Dallas, TX), anti-FSP-1 (Abcam), anti-CD31 (Abcam), anti-VE-Cadherin (Abcam), anti-α-SMA (Sigma-Aldrich).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology); was used as loading control. Thereafter, blots were incubated with HRP-labeled respective (anti-mouse or anti-rabbit) secondary antibodies (Santa Cruz Biotechnology), washed and processed with Clarity™ Western ECL Substrate (Thermo Fisher Scientific). Signal was detected using ChemiDoc™ Touch image analyzer (Bio-Rad Laboratories).

Total protein from EndMT-undergoing HUVECs was extracted and size-fractioned by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk in PBS with 0.1% Triton-X100, immunodetection was carried out using specific primary antibodies: anti-p-p65 (Cell Signaling, Danvers, MA), anti-p65 (Santa Cruz Biotechnology), anti-p-Smad2 (Cell Signaling), anti-Smad2 (Cell Signaling), anti-p-p38 (Cell Signaling), anti-p38 (Cell Signaling), anti-p-Erk (Cell Signaling), anti-Erk (Cell Signaling), anti-TWIST (Santa Cruz Biotechnology), anti-FSP-1 (Abcam), anti-CD31 (Abcam), anti-VE-Cadherin (Abcam), anti-α-SMA (Sigma-Aldrich), anti-E-Selectin (Santa Cruz Biotechnology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology); was used as loading control. Thereafter, blots were incubated with HRP-labeled respective (anti-mouse or anti-rabbit) secondary antibodies (Santa Cruz Biotechnology), washed and processed with Clarity™ Western ECL Substrate (Thermo Fisher Scientific). Signal was detected using ChemiDoc™ Touch image analyzer (Bio-Rad Laboratories).

The cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected and denatured. Proteins were separated by 10% SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were treated with TBST containing 50 g/l skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies, anti-KLF4 (Cat. no. BM0485; Abzoom Biolabs, Dallas, TX, USA), anti-E-cadherin (Cat. no. BM0530; Abzoom Biolabs), anti-α-SMA (Cat. no. YT5053; ImmunoWay Biotechnology Company, Newark, DE, USA), anti-ZO-1 (1:1,000 dilution; Cat. no. ab59720; Abcam), anti-FSP-1 (1:5,000 dilution; Cat. no. ab27957; Abcam) and anti-β-actin (Cat. no. BM0272; Abzoom Biolabs), respectively, at 37°C for 1 h. The membranes were rinsed and incubated for 1 h with the corresponding peroxidase-conjugated secondary antibodies. Chemiluminescent detection was performed using the ECL kit (Pierce Chemical Co., Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of β-actin.

Immunostaining was performed in 4-well chamber slide. After fixing with 4% paraformaldehyde for 10 min and washing with PBS, blocking solution (5% bovine serum albumin in PBS with 0.1% Triton-X100) was applied for 1 h. Primary antibodies (anti-CD31: Abcam, anti-FSP-1: Abcam, anti-VE-Cadherin: Abcam and anti-α-SMA: Sigma-Aldrich) were applied overnight at 4 °C. Cells were washed with PBS, and corresponding fluorescence-tagged secondary antibodies were applied for 1 h at room temperature. After washing, the cells were mounted using VECTASHIELD^TM anti-fade mounting medium with DAPI (Vector laboratories). Immunostaining was observed under Olympus Fluoview FV200i confocal fluorescent microscope (Olympus).

5000 fibroblasts were seeded per well in a 96 well plate in DMEM/10% FBS overnight. Cells were fixed in 10% neutral formalin buffer for 24 hours, permeabilized in methanol for 10 minutes at -20°C and blocked in PBS containing 3% FBS for 1 hour. Cells were then incubated with the following antibodies (1:100) for 24 hours in blocking buffer: anti-PDGFR-α (Cell Signaling Technology, cat no.5241), anti-FSP1 (Abcam, cat no. ab75550), α-SMA (Abcam cat no. 7817), anti-VEGFR2 (Santa Cruz Biotechnology, cat no.sc-393163) and anti-Pan-Cytokeratin (Biolegend, cat no.628602). Cells were washed in PBS 3 times and incubated with the following secondary antibodies (1:1000): anti-rabbit-biotinylated (Jackson Laboratories, cat no.111-065) to detect PDGFR-α or anti-mouse-biotinylated (Vector Laboratories, cat no.BA-9200) to detect VEGFR2, α-SMA or Pan-Cytokeratin. Biotinylated antibodies were incubated with streptavidin bound to horseradish peroxidase (HRP) (Vector Laboratories, cat no. SK4100) for 30 minutes. FSP1 was detected using secondary anti-rabbit-HRP (1:500, Avantor, cat no. 10150-732). Protein expression was detected through HRP reaction to 3, 3 -diaminobenzidine substrate (Vector Laboratories, cat no.SK4100).