Goat anti rabbit igg h l

For immunohistochemistry, the dish climbing glasses of cells were collected and fixed with 4% paraformaldehyde solution for 20 minutes at 4 °C. These samples were washed 3 times by PBS and then incubated with normal goat serum overnight. After being immersed in the diluted primary antibody for 2 h, they were incubated at 4 °C with the second antibody. The antibodies used in this experiment were anti-USP37 (1:500, 18465-1-AP) and goat anti-Rabbit IgG (H+L) (1:200; SA0000I-2) purchased from Proteintech Group Inc. Representative images were photographed and positive cells in the images were stained brown.

Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (STEMCELL Technologies, Kent, WA, USA), according to the manufacturer’s instructions. PBMCs and EVs were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS] in Tris-buffered saline) with protease inhibitors on ice for 30 min. Proteins obtained from EVs and PBMC were electrophoresed in 10% SDS-polyacrylamide gels and then transferred to 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk for one hour at room temperature. After probing with primary antibodies at 4 °C overnight, the membranes were incubated by HRP-conjugated secondary antibodies (goat anti-Mouse IgG (H + L), goat anti-Rabbit IgG (H + L), Proteintech). The antibodies used were anti-CD63 (Proteintech, 25682-1-AP), anti-TSG101 (Proteintech, 14497-1-AP) and anti-Calnexin (Proteintech, 10427-2-AP), goat anti-Mouse IgG (H + L) (Proteintech, SA00001-1) and goat anti-Rabbit IgG (H + L) (Proteintech, SA00001-2). The detection of immune complexes was performed using a LumiBest ECL Reagent Solution kit (Share-Bio, Shanghai, China).

TOPK (sc-293028) was purchased from Santa Cruz Technology, Inc (Santa Cruz, CA). Flag (F1804) was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to detect ALK (#3633), p-ALK (#3341), p-JNK (#4668), JNK (#9258), p-ATF2 (#5112), ATF2 (#9226), PARP (#9542), c-PARP (#5625), HA (#3724), p-Tyrosine (#8954) were purchased from Cell Signaling Technology (Danvers, MA). β-actin (66009-1-Ig),α-Tubulin (66031-1-Ig), HRP-labeled Goat anti-Mouse IgG (H + L) and Goat anti-Rabbit IgG (H + L) were from Proteintech Group, Inc (Wuhan, HB). Phospho-TOPK at Y74 antibody was prepared by Abgent, Inc (Suzhou, JS). All antibodies were used following the instructions of the respective manufacturers.

Antibodies were incubated with Dynabeads protein G (Invitrogen, Carlsbad, US) at 4°C for at least 30 minutes. Cells were lysed in NP-40 buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (Thermo Fisher, Waltham, US). Cell lysate was incubated with antibody-coated beads at 4 °C overnight. Antibodies used for immunoblotting and co-immunoprecipitation included anti-Vam6 (Thermo Fisher, Waltham, US), anti-VDAC1 (Abcam, Cambridge, UK), anti-Rab7a (NewEast, Kelayres, US), anti-AMPKγ (Invitrogen, Carlsbad, US), anti-mCherry (Invitrogen, Carlsbad, US), anti-β-actin (Proteintech, Chicago, US), and rabbit IgG isotype ctrl (Invitrogen, Carlsbad, US). HRP-conjugated secondary antibodies included mouse-anti-rabbit IgG light chain (CST, Danvers, US), goat-anti-mouse IgG (H+L) (Proteintech, Chicago, US), and goat-anti-rabbit IgG (H+L) (Proteintech, Chicago, US). Antibodies and isotype controls used in co-immunoprecipitation experiments were at 1 μg/mL, and antibodies for immunoblotting were used at 1:1000 dilution unless other described.

For immunohistochemistry, slides were preincubated with 3% H₂O₂ for 10 minutes. Goat serum was used to block the nonspecific binding, and then, sections were incubated overnight at 4°C with rabbit anti‐cathepsin K polyclonal antibody (Abcam, Cat#ab19027) or rabbit anti‐Osx polyclonal antibody (Abcam, Cat#ab209484). Subsequently, sections were incubated with horseradish peroxidase‐conjugated secondary antibody for 30 minutes at 37°C. Colour was developed using DAB substrate kit (Boster, Cat#13J25J14J1022), and haematoxylin was used for counterstaining. The number of positive cells per unit area in the distal socket was calculated.
For immunofluorescence staining, the slices were blocked with 5% BSA and then incubated overnight at 4°C with a rat anti‐CD90 polyclonal antibody (BioLegend, Cat# 105201), a rat anti‐CD44 polyclonal antibody (BioLegend, Cat#103003), goat anti‐Osx monoclonal antibody (Santa Cruz, Cat# sc‐393325) and a rabbit anti‐CTSK polyclonal antibody (Abcam, Cat#ab19027). After that, the sections were incubated with anti‐rat IgG (H + L) (Proteintech, Cat# SA00003‐11), donkey anti‐goat IgG (H + L) (R&D, Cat#F0108) and goat anti‐rabbit IgG (H + L) (Proteintech, Cat#SA00013‐2) for 30 minutes. Nuclei were counterstained with DAPI. Images were captured on a Nikon A1plus confocal laser scanning microscope.

The JBMMSC were seeded on the glass slides until 80% confluence. Then Lyso Tracker (KeyGEN, Cat#KGMP006) was added and incubated with cells at 37°C for 1 hour. After that, cells were fixed in 4% paraformaldehyde. Cells were permeabilized in PBS containing 0.5% Triton X‐100 for 20 minutes and blocked with normal goat serum for 30 minutes at room temperature. Then, the cells were incubated overnight at 4°C with a rabbit anti‐CTSK polyclonal antibody (Abcam, Cat#ab19027). After that, the sections were incubated with goat anti‐rabbit IgG (H + L) (Proteintech, Cat#SA0013‐2) at 37°C for 30 minutes. Nuclei were counterstained with DAPI.

The total protein of serum exosomes, placental villous exosomes, placental villous tissues and cells was isolated using the RIPA Lysis Buffer (Beyotime) and quantified by the BCA Protein Assay Kit. Samples were boiled at 100°C for 5 min in 5× sample loading buffer, separated on 8–15% SD‐polyacrylamide gel, and transferred to 0.22 or 0.45 μm PVDF membranes. Membranes were blocked with 5% skim milk for 2 h or QuickBlock™ Blocking Buffer (Beyotime) for 20 min at room temperature (RT) and incubated with primary antibodies overnight at 4°C. Then, samples were incubated with the appropriate secondary antibody labelled with horseradish peroxidase. Chemiluminescent signals were detected using Femto‐sig ECL Western Blotting Substrate (Tanon, China). Images were captured using a chemiluminescence imaging system (Bio‐Rad USA). Densitometric protein level analysis was performed using Image Lab. Primary antibodies against E‐cadherin (Cat# 20874‐1‐AP), N‐cadherin (Cat# 22018‐1‐AP), vimentin (Cat# 10366‐1‐AP), GAPDH (Cat# 10494‐1‐AP) and the secondary antibody Goat Anti‐Rabbit IgG (H + L) (Cat# SA00001‐2) were purchased from Proteintech (USA); primary antibodies against p38 MAPK (Cat# 8690 T) and Phospho‐p38 MAPK (Cat# 4511 T) from Cell Signaling Technology (USA); primary antibodies against CD6 (Cat# ab193349), CD81 (Cat# ab83) and TSG101 (Cat# ab109201) from Abcam (UK).